European Journal of Pharmacolol,% 213 (1992) 141 - 144
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
External ATP antagonizes the effect of potassium channel openers in guinea-pig ventricular papillary muscle A n d r e a B o t t ;', M a n f r i d E l t z e ;' a n d P e t e r llles ~,b " Department of Pharmacology, Byk Gulden Pharmaceuticals, tJvk-Gulden-Strasse 2, I)-7750 Konstanz, German), and h l)epartment of Pharmacology, Unit'ersity of Freiburg, Hermann-Herder-Strasse 5, D-7800 Freiburg, Germany Received 3 January 1992. accepted 21 January 1992
Right ventricular papillary muscles of the guinca-pig heart were electrically stimulated. Cromakalim 10-100 >M and Ro 31-6930 3 tzM depressed the contractile forcc and shortened the duration of action potentials. Glibenclamide 0.3-3 >M, ATP 100 ,o.M and a,/3-mcthylcnc ATP (a,/3-mcATP) 30 #M antagonized these cffccts. Suramin 300 p.M failed to reverse the o~,,8-mcATP-evoked antagonism of thc action of cromakalim. I1 is concluded that both intra- and cxtraccllular ATP may interfcrc with potassium channel openers and that cxtraeellular ATP does not act via the known P2-purinoceptor subtypes. Ventricular papillary muscle; Contractile force; Action potentials; K ~ channel opcncrs; Sulfonylurca antidiabetics; P2 purinoceptor agonists
A new type of K ÷ c h a n n c l that is inhibited at physiological intracellular A T P concentrations has recently been described in the heart (Noma and Takano, 1991). Under hypoxic conditions the A T P content decreases and the channel opens; the resultant outflow of K* is thought to be responsible for the shortening of the action potential and the decreased inotropy. In addition to metabolic exhaustion, a certain class of drugs, the so-called potassium channel openers (e.g. cromakalim), also activate the ATP-sensitive K ÷ current (IK(ATF,)) ( C o o k , 1988). Sulfonylurea antidiabetics, such as glibenclamide, decrease I K(ATP) and thereby antagonize the effects of potassium channel openers. Both extracellular A T P and its degradation product adenosine may cause changes in myocardial contractility by activating m e m b r a n e purinoceptors of the P2and Pl-type, respectively (Burnstock and Kennedy, 1986). In guinea-pig atria, only adenosine has a negative inotropic effect, while in guinea-pig ventricles both adenosine and A T P are inactive (Burnstock and Meghji, 1981). Since external ATP has been shown to inhibit 1K(ATP) in an insulin-producing cell line ( A r k h a m m a r et al., 1990), we studied, in guinea-pig papillary muscle, whether A T P itself or the enzymatically stable struc-
Correspondence to: P. llles, Department of Pharmacology, University of Freiburg, F.R.G. Tel. 49.761.203 3465, fax 49.761.203 4235.
tural analogue a,fl-methylene A T P (a,/3-mcATP) affects thc actions of potassium channel openers on I K(ATP)'
2. Materials and methods
2.1. Preparation and recording Male guinea-pigs (300-500 g) were killed by cervical dislocation. Right ventricular papillary muscles ( < lmm diameter) were prepared and set up as described (Galvan and Schudt, 1990). Briefly, the muscle was perfused in an organ bath with medium at a rate of 10 m l / m i n . The medium ((mM): NaCI 118, KCI 3.4, N a H C O 3 25, K H 2 P O 4 1.2, MgSO 4 1.2, CaCI 2 2.4, glucose 5.5 and Na-pyruvate 2) was saturated with 95% 0 2 - 5 % CO 2 and maintained at 34°C. Muscles were stimulated (Stimulator T, Hugo Sachs) with 1-ms square-wave pulses, via a pair of platinum wires placed immediately under the tissue. The stimulation strength was about 10% above threshold voltage (2-15 V) and the stimulation frequency was 0.2 Hz. The tendon of the papillary muscle was connected to an isometric force transducer (Statham UC2) and the preload was set to 400 mg. Potentials were recorded intracellularly with microelectrodes filled with 3 M KC! (15-25 M.f2 tip resistance) and connected to a high-impedance voltage follower (Axoclamp II). After amplificatkin, the potentials and the force of contraction were
sampled, stored and analyzed by a computer (IBM XT-286) fitted with an AD converter (Metrabyte, DASH 16F).
2.2. Application of drugs and evaluation of data
We measured, in 81 papillary muscles obtained from guinea-pig right cardiac ventricles, the electrical and mechanical responses to stimulation with l-ms squarewave pulses delivered at a frequency of 0.2 Hz. The contractile force amounted to 80.4 + 6.0 mg. The resting membrane potential was - 8 6 . 7 + 0.4 mV and the action potential had an amplitude of 124.5 + 0.4 mV and a duration of 162.9 + 3.5 ms. Even the highest concentrations of cromakalim (30 ~M) and Ro 31-6930 (3 /xM) failed to later the membrane potential; at the same time the amplitude of action potentials was slightly decreased ( < 7%) (for cromakalim see fig. 1). All other substances were completely ineffective on these parameters.
Muscles were allowed to equilibrate for about 1 h before the experiment was started. After successful impalement, the contractile force and the intracellular potentials were measured at regular intervals of 5-10 min. Wc determined the mcmbranc potential, as well as the amplitude and duration (at 50% amplitude) of the action potentials. After collection of control data for 10 min, increasing concentrations (3-100 /zM) of cromakalim were applied cumulatively. The contacttime of each concentration was 20 min. In another series of experiments, cromakalim 30 /zM (or Ro 316930 3 /xM) was applied for 20 min (T I) and subsequently washed out. After an additional 60 min, the same concentration of cromakalim (or Ro 31-6930) was reapplied for 20 min (T2). In subsequent experiments, 10 min before and during T 2, ATP 300 tzM or a,/3-meATP 3-30 ~zM was also present in the medium. In some cases, a,/J-meATP 30 # M was added together with suramin 300 /zM. The percentage inhibition (T~ and T 2) produced by the potassium channel openers was calculated at the maximal effects 20 min after application. T 2 is expressed as a percentage of T v
3.1. Parameters determined
3.2. Effect of cromakalim and interaction with glibenclamide Cromakalim concentration dependently inhibited both the contractile force (28.2_+ 5.5% at 10 p,M, 74.2_+ 5.1% at 30 p~M and 89.2_+ 4.0% at 100 /zM; n = 14) and action potential duration (12.8 +_ 4.2% at 10 gM, 76.1 _+ 2.3% at 30 ~ M and 95.2_+ 2.1% at 100 /xM; n = 14). As shown in another series of experiments, the effect of cromakalim 30 ~ M was completely
2.3. Drugs The drugs used were suramin (E. M611er, Bayer, Wuppertal, FRG); 2-(6-cyano-2,2-dimenthyl-2H-l-benzopyran-4-yl)pyridine-l-oxide (Ro 31-6930; D. Flockerzi, Byk Gulden, Konstanz, FRG); glibcnclamide (H. Englert, Hoechst, Frankfurt am Main, FRG); adenosine 5'-triphosphate disodium salt (ATP), a,/3-methyieneadenosine 5'-triphosphate lithium salt (a,/3meATP; Sigma, Deisenhofen, FRG); cromakalim (S. Patak, Smith, Kline & Beecham, Betchworth, GB). Stock solution (1-100 mM) of all compounds were prepared in the following solvents: distilled water (ATP, a,/3-meATP, suramin), 70% ethanol (cromakalim, Ro 31-6930) and 0.1 N NaOH (glibenclamide). Further dilutions were made with medium. Equivalent quantities of the solvents had no effect.
2.4. Statistics Means + S.E. are given throughout. The KruskalWallis analysis followed by the Mann-Witney test was used for comparison of the means. A probability level of 0.05 or less was considered to be statistically significant. In the case of multiple comparisons, the significance level was adjusted according to the method of Bonferroni.
( It 0~.t
] 0 IT~
( t 18 + ( RO~.~
Fig. 1. Effect of cromakalim and its interaction with glibenclamide in guinea-pig right papillary muscles. Stimulation was with square-wave pulses of 1 ms duration, a frequency of 0.2 Hz, and a voltage about 10% above threshold. (A) After recording the contractile force (upper trace) and action potential (lower trace) under control conditions (CONT), cromakalim 30 ~ M was applied for 20 min (CROM). (B) After subsequent washout of cromakalim 30 H.M, an additional 50 rain was allowed to elapse (CONT). Glibenclamide 3 # M was then applied for 10 rain alone (GLIB) and for an additional 20 rain together with cromakalim 30 g M ( G L I B + C R O M ) . Calibrations in (A) and (B) are identical.
-~ ~ I°° 25 1
I ~ . RO
()(ll 0 ~ I (;IIB
Fig. 2. Effects of potassium channel openers as well as their interaction with glibenclamide and P2-purinoceptor agonists. The membrane potential (upper panel) and the duration of the action potential (at 50% amplitude; lower panel) were measured. After collection of control data for 10 min, cromakalim 30 `aM (CROM) or Ro 31-6930 3 ,aM (Ro) was applied for 20 min (T I) and subsequently washed out. After an additional 60 min the same concentration of cromakalim or Ro 31-6930 was reapplied for 20 min (T:). In further experiments, 10 min before and during T 2, ATP or a,fl-meATP (meATP) was also present in the medium. In some cases, a,/J-meATP was added together with suramin (SUR). The percentage inhibition (T I and T,) produced by the potassium channel openers was calculated at the maximal inhibition, 20 min after application. T, is expressed as a percentage of T t. The concentrations of drugs are indicated in ,aM. Each column depicts the mean + S.E. of six experiments. * Significant differences (P < 0.05-0.01) between the effect of cromakalim in the presence and absence of glibenclamide (first set of columns) or purinoceptor agonists (second set of columns); significant differences between the effect of Ro 31-6930 in the presence and absence of a./3-meATP (third set of columns). There was no significant difference (P > (I.05) between the effect of cromakalim in the presence of r~,,(J-meATP,and a,/3-meATP plus suramin.
reversible on washout ( c o m p a r e figs. 1A and 1B) and was fully reproducible after 60 min (fig. 2). Figure 1B shows that glibenclamide 3 /.zM did not cause a major change in the mechanical or electrical responses, but abolished the effect of cromakalim 30 p.M. W h e n all experiments were taken into consideration, glibenclamide on its own had no activity either at 0.3 or at 3 p.M. However, both concentrations of glibenclamide strongly antagonized the effect of cromakalim 30 /~M; glibenclamide 0.03 /~M was completely inactive (fig. 2).
3.3. Interaction o f A T P and makalim or R O 31-6930
than glibenclamide (fig. 2). The antagonistic effects of A T P and a , / 3 - m e A T P reached significance only when changes in action potential duration, but not when changes in contractile force were evaluated (fig. 2). R o 31-6930 3 g M p r o d u c e d an inhibition similar to that of cromakalim 30 /xM. O n c e again a , / 3 - m e A T P 30 g M antagonized the effect of R o 31-6930 3/,tM only on the duration of action potentials (fig. 2). Thus, a higher concentration (30 ~ M ) of a , / 3 - m e A T P was clearly antagonistic against the effects of both cromakalim 30 /,LM and Ro 31-6930 3/.tM, while a lower concentration (3 ~ M ) of a , / 3 - m e A T P was inactivc.
c~,fl-meATP with cro-
W h e n given alone, A T P 100 /sM and a , / 3 - m e A T P 3 - 3 0 /.tM were without effect on the contractile force and the duration of action potentials. However, in interaction experiments, both A T P 100 # M and a,/3m e A T P 30 /.LM r e d u c e d the depression induced by cromakalim 30 /xM, although with a lower potency
3.4. Failure o f suramin to alter the interaction between cr,fl-rneA TP and cromakalim Suramin 300 /zM on its own did not change the mechanical or clectrical responses. Moreover, it failed to influence the interaction between a , / 3 - m c A T P 30 /.tM and cromakalim 30 ~ M (fig. 2).
4. Discussion Cromakalim, the prototypic potassium channel opener, is a racemic benzopyran derivative; R o 31-6930 has a related non-chiral structure of much higher biological potency (Edwards and Weston, 1990). As described repeatedly for this class of drugs (see i.e. Sanguinetti et al., 1988), cromakalim and R o 31-6930 had a negative inotropic effect in guinea-pig papillary muscles. They decreased the duration but did not alter appreciably the rate of rise or amplitude of action potentials. The resting m e m b r a n e potential was not c h a n g e d either. In accordance with previous results (Sanguinetti et al., 1988), the cardiac effects of potassium channel o p e n e r s were antagonized by the sulfonylurea antidiabetic glibenclamide. Glibenclamide on its own was inactive, since u n d e r physiological conditions A T P - s e n sitive K + channels in the heart are closed at the resting m e m b r a n e potential. T h e situation is different u n d e r pathophysiological conditions. Blockade of oxidative phosphorylation leads to a decrease in intracellular A T P and, thereby, to activation of IK(ATP) (Fossct et al., 1988). T h e ensuing shortening of the action potential is antagonized by glibenclamide. A T P and its structural analogue a , f l - m e A T P had no effect on the electrical or mechanical properties of guinea-pig papillary muscles. Similar findings have been reported for the atria and ventricles of this species (Burnstock and Meghji, 1981). However, both comp o u n d s antagonized the depression of action potential duration caused by cromakalim or R o 31-693. Glibcnclamide and a , f l - m e A T P shifted the c o n c e n t r a t i o n - r e sponse curve of cromakalim in a parallel m a n n e r to the
right, but did not alter the concentration-response curve of the Ca 2+ entry blocker nitrendipine (A. Bott, M. Eltze and P. llles, unpublished observation). Thus, the interaction between cromakalim and glibenclamide (or a,/3-meATP) appears to be selective and is compatible with competitive antagonism. Since a,/3-meATP is much more resistant to dephosphorylation than ATP, an indirect action at Pt-purinoceptors, via the generation of adenosine, is unlikely. Instead, the effects of ATP and a,/3-meATP may be due to the stimulation of a P2-purinoceptor which is suramin-insensitive and therefore does not belong to the known subtypes P2x or P2Y- In our experiments, 300 /~M ATP and 30 # M a , ~ - m e A T P were used; these are rather high concentrations but comparable to those required for the activation of P2-purinoceptors in various tissues. In cardiac myocytes, an elevation of intracellular ATP inhibits ATP-sensitive K ÷ channels (Noma and Takano, 1991) and, in addition, counteracts the activation of I K(^TP)by potassium channel openers (Thuringer and Escande, 1989). Recently, extracellular ATP has also been shown to inhibit I K(ATr') in an insulin-producing cell line (Arkhammar et al., 1990). We now document that, in guinea-pig papillary muscles, external ATP interacts with both cromakalim and Ro 31-6930. Since glibenclamide antagonizes the opening of the same class of K ÷ channels during hypoxia or metabolic exhaustion, an effect inhibited by intracellular ATP (Fosset et al., 1988), it may be assumed that extracellular ATP has a similar effect. In fact, under these pathophysiological conditions large quantities of ATP leave the heart muscle ceils and may activate cardiac Pz-purinoceptors. This results in a restitution of the previously shortened action potentials.
Acknowledgement The generous gift of drugs by Bayer, Byk Gulden, Hoechst and Smith, Kline & Beecham are gratefully acknowledged.
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