Inotropic effect of amrinone in rabbit papillary muscle: reversal of the myocardial depressant effect of halothane'

HIKWHIMA KOMAI~ AND BEN F. RUSY depart men^ ~fAlaestBaesd~lo,qy, Canter JkPr Health Sciences, Uraiversity ojg. Wisconsin, Madison, WB, U.S,iB. 53792

Received March 6. 1984

KOMAH, H , , and B. F. RUSY. 1984. Inotropic effect of aminone in rabbit papillary muscle: reversal of the myocardial depressant effect of halothane. Can. %. Physiol. P h m a c o l . 62: 8 382 - 1386.

hn rabbit papillary muscles, the pronounced inotropic effect of aminone (0.4-0.8 m g / d ) observed at low stimulation frequencies (0.1 -0.5 Hz) diminished as the frequency was increased to 1.0-2.0 Hz. Reversal of the negative inotropic effect of halothane (1%) by aminone 40.8 mg/ml) was similarly dependent on stimulation frequency. The contractile response to paired-pulse stimulation in the presence of aminsne (0.8 rng/ml) was summation rather than postextrasystolic potentiation. Aminone 40.8 m g / d ) was not effective in reversing the negative inotropic effect of halothane (1%) on potentiated state contraction generated by paired-pulse stimulation. These results suggest that amrinone increases the influx of extracellular Ca2+ across the sacoiemrna but not the availability sf cellular (la2* which accumulates, most likely in sarcoplasmie reticulum, under the condition of potentiation.

KOMAI, H.. et B. F. Rus'f. 1984. Bnotrslpic effect of arnrinone in rabbit papillary muscle: reversal of the myocardial depressant effect of halothane. Can. J. Physiol. Phamacol. 62: 1382- 1386.

Dans les muscles papillaires de lapin, l'effet inotrope prowoncC de l'aminone ((b,4-0,8 m g / d ) observe B des frkqalences de stimulation faibles (0,1 -0,5 Hz) diminua B mesure que I'on augmenta la frCquence 2 B ,68-2,0 Hz. LC renversement de l'effet inotrope nkgatif de 19halothane (1%) par l'aminone (0,X rng/mk) fut similarirement fonctisn de la frCquence de stimulation. La rkponse contractile h uwe stimulation d'impulsions pair&es en presence d'aminone (0,8 ~ng/mL) fut davantage une somrnation qu'une potentialisation post-extrasystoliq~c~ L'amP-inone (0,8 mg/mLB fut incapable de renverser l'effct inotrope &gatif tie i'halobhane (1 %) sur Ia contraction de l'ktat potentialid induite par Ia stimulation d'impulsions pairks. Ces dsultats sraggkrent que I'amrinone augmente I'influx de Ca" extracellulaire 3 travers le sarcolemme mais non la disponibilit6 du ~ a " cellulake qui s'accumule, plus vraisemblablement dans le rkticulurn sarcoplasmique, pendant la potentialisation.

[Traduit p a le journal]

Aminone is a noneatecholamine, nonglysosidic inotropic agent which has been shown to inhibit phosphodiesterase and to increase the cAbfP content of myocardium (Honerjiiger e6 al. 1981; Endsh et a&. 1982). The mechanism underlying its posi- tive inotropic effect is still not well understood. It is wow generally accepted that rnyoczardial contractile activity depends on the availability of cellular Ca'2 as well as on the influx of extracellular Cai (Morad and Goldman 1973). How an ino- tropic agent influences each of these sources sf Cat2 is am- portant to a detailed understanding sf its mechanism of action. Since an increase in stimulation frequency most likely in- creases the influx of extracellular Ca+2 (Edrnan and J6hannsson 19%6), and potentiating conditions such as paired-pulse stirnu- latisn most likely increase the contribution of iwtracellulararly stored Ca" (Bass 1976; Sutko eb a&. 1979), we have studied the effect sf amrinone under different stimulation frequencies as well as under potentiating conditions to gain insight into the effect of amlrawone ow the availability of the two different sources of Cae2. We have also evaluated aminowe's ability to counteract the negative iwotropic effect of Baalothane, which appears to suppress the availability of both extracellular and intracellular Ca ' ' (Komai and Rusy 1982'1.

Materials and methods Rabbits (about 2 kg) were anesthetized by intravenous injection of

pentobarbital (approximately 45 mg/kg). The heart was excised and one papillary muscle from the right ventricle was isolated from each h e m . The muscle was mounted vertically in a tissue bath (45 d)

maintained at 30°@. The lower end of the muscle was tied to a fixed base and the upper end to a stainless steel wire attachcd to a Statham UC-2 force transducer. Hsomegric developed force was recorded on a @i%sean polygraph. The length and cross-sectional area of the muscles were obtained .from measurements of muscle length and diameter by an ocular micrometer using a Unitron binocular microscope. The dimensions (mean .C SD) of the 23 muscles rased in this study were length, 3 -9 9 1.0 mm and cross-sectional area, 0.70 5 0.3 1 mm2. The force was expressed in units of rnilliNewtons per square rnillimetrc of cross-sectional area of a muscle. The muscIes were stimulated at 0.1 Hz for 2-3 h to allow stabilization. The stimuli (4 ms duration, 1.5 x threshold) were delivered through silver-silver chloride field elec- trodes. A laboratory stimulator (American Electronic Laboratories, model 104 A) was used. The resting tension was maintained at BO mhi in all the experiments. The superfusing medium was a modified bebs-Henseleit bicarbonate (pH '7.4) of the following composition; NaCI , 1 15 mM; KC], 5.9 mM; MgCl,, 1.2 mM; NaH2PQ4, B .2 I ~ M ; Na,SC149 1.2 mM; NaHCO,, 25 mM; CaCI2, 2.5 mM; ethyl- enediarninetetraacetate (EDTA), 58 pM; and glucose, 5.6 mM- Am- rinone was dissolved in an equivalent concentration of HCI. The final pH of the n~cdium containing up to 0.8 m g / d of aminone was only about 0.1 unit below that of control medium. The medium was equil- ibrated with a gas mixture of 95% O2 and 5% 6102 by continuous bubbling. When the effect of halothane was studied, the anesthetic was added to the above gas Pafixture using a calibrated FIuotec vapor- izer. A stable force of contraction at a given stimulation frequency thoughout the experimental period was used as the criterion for effec- tive equilibration following addition of drugs. Rested state con- tractions were elicited by stimulating muscles after a 20-min rest period without stimdatisn. The muscle preparations were found to be stable throughout the duration of experiments which lasted approxi- mately 4 h. The results were statistically evaluated by Student's paired or unpaired f-test as indicated.

% preliminary account of this work was reported at the annual meetings of the American Society of PharrnacoIogy and Experinsental Therapeutics, New Orleans, April 1982 and Chicago, April 1983.

"uthor to whom all correspondence should be addressed. A positive staircase was demonstrated in the control condi-

tion for two separate groups of muscles (Fig. I , n .= 6 ; Fig. 2,

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KOMAI AND WUSY I383

F R E Q U E N C Y ( H z )

FIG. 1. Inotrogic effect of amsinone at various stimulation fre- quencies. Means k SEM of dctem~inations in six musclcs are shown. Amrinone (Am) concentration (milligrams per rnillilitre) as indicated. *, p < 0.05 compared with comespondirag control value (paired t-test).

n -- 8). Unpaired t-test showed no statistically significant dif- ferences between conesponding data points of these two groups. The positive inotropic effect of arnrinone at coneen- trations of 0.4 and 0.8 rng/mL was strongly dependent on the frequency of stimulation (Fig. 1) and the pronounced effect observed at Isw stimulation frequencies diminished as the fre- quency was increased. At a stimulation frequency of 2.0 Hz, developed force in the presence of aminone was not different from control. The effect of aminone was similar in both groups (Figs. 1 and 2) and, for the 0.8 mg/ml concentration, unpaired t-test showed no significant differences between corresponding data points. For both concentrations of arnrinone t h e forceL frequency relationship tended to flatten and the accumulation of frequency inotropy was shifted to a lower frequency range. Since an unequivocal effect of arnglnone was sought in the present study, a csncentration of 0.8 rng/rnL was used in the subsequent experiments. This concentration of the drug is com- parable to the concentrations bland by others to have a pro- nounced positive inotropic effect in isolated papillary muscles (Aloaasi ed al. 1979; Onuaguluchi and Tanz 1981 ; Honerjiiger et a1. 1981).

The frequency dependence of the inotropic effect of am- rimonc persisted when the medium contained halothane, as 0.8 mg/m& of aminone effectively reversed the negative inotropic effect of 1 % halothane at low but not at high stimulation fre-

FREQUENCY ( H E ) ;

FIG. 2. Effcct of amrinone, halothane, and amrinone plus halo- thane on the developed force at various stimulation frequencies. Means 2 SEM of determinations in eight muscles are shown. *, p < 0.05 compared with corresponding csntroI value (paired t-test). Halo- thane (Hal), 1% in gas phase. Anxinone (Am), 0.8 rng/mL.

TABLE I . Effect of habthane, arnrinone, and halothane plus am- finone on rested state contraction

Control 3.920.S + Halothane ( 1%) 2.0zk0.3* + Amriwone (0.8 rng/rnL) 4.5+0.8* +Halothane ( I % ) + aminone (0.8 rng/wP&) 3.4zk0.04

NOTE: Values are mean & SEM (ra = 8). *g < 0.01 csmpxcd with control (paired t-test).

traction, and effectively reversed the negative inotropic effect of halothane ( I % ) on the rested state contraction.

Figure 3 illustrates the patterns s f the contractile: responses of the muscles to paired-pulse sti~nulation under the control condition and in the presence af arnrinone (0.8 rng/mL.). For eight rnuscles under the control condition, the developed force (mean + SEM) of the second of the pair of peaks was 58 t 4% sf that of the first peak. In the presence of 0.8 ing/rnl am- finone. the developed force of the second peak was 4 18 & 7% of that of the first peak. 'barns, in the presence of aminone, paired-pulse stimulation resulted in summation rather than postextrasystolic potentiation.

As can be seen in Fig. 4, 1% halothane inhibited the force quencies (Fig. 2 ) . As can be seen in ~ a b l r I , amrinone (0.8 of contraction generated by single-pulse (0.2 Hz) stimulation. mg/mE) enhanced the developed force sf the rested state con- Paii-ed-pulse stimulation resulted in postextrasystolic poten-

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H 384

C O N T R O L

CAN. 9. PHYSIOL. PWARMACOC. VOL. 62, B9d4

RG. 3. Typical patterns of contraction elicited by paired-pulse stimulation under the control condition and in the presence of aminone. The basal frequency was 0.2 Hz. The record (left to right) shows the contractile responses sf a muscle to single-pulse stimulaticsn (first three beats) and ts paired-pulse stimuiatisn with time intrapair coarpling interval of 300 ms (fourth and subsequent beats).

tiation in the presence of halothane, although halothane in- hibited both the potentiated state contraction (first peak) and the extrasystole (second peak). Aamrinone (0.8 rng,irnL) effectively counteracted the negative inotropic effect of !lalothane ( B %/c) during single pulse (0.2 Hz) stimulation. It also effectively counteracted the negative inotropic effect of halothane ow the second peak but not the first perk generated by paired-pulse stimulation. In the absence of halothane, arminone signifi- cantly enhanced the force of c~n&actiogl generated by single- pulse stimulation and both peaks generated by paired-pulse stimulation. The response of muscles to paired-pulse stimu- lation in the presence sf atminone was sulrnmation (P2 > PI) rather than postextrasystolic potentiation, regardless sf the presence or absence of halothane.

The observed frequency dependence of the positive instropic effect of aminone suggests an action analogous to that of an increase in stimulation frequency, since the effect of amriglone was rninjlmal at high stimulation frequencies where this form of inotropy is already close to maximurn in the absence of the drug. The action is similar to that caused by srcip~enaline (Hilgernann et a&. 1974) in that it shifts the accurnulatic?n of frequency inotropy to a lower frequency range, an effect which is suggested to occur with kca-adrenergic agowists, phss- phodiesterase inhibitors, and CAMP analogues (Mensing and Hilgennann 1981). Such an effect has been denaonstrated for caffeine (Henderson 6.f a/ . 4974) as wclH as for high csncen- trations of extracellular Ca2' , mcthoxamiwe, (or low concen- trations of isoproterenol (Siegl and McNeill 1982). Note that halothane serves only to reduce the rmaximurn of frequency inertropy accumulation and that it does not shift the range in which frequency inotropy occurs either in the control condition (3% in the presence sf aminone. This effect of halothane is similar to that found for drugs which act by a CAMP-inde- pendent means such as nifedipirae. adenssine, and decreased extracellular Ga" concentration (Mensing and HiHgemann 1981).

Increase in the stimulation frequency increases the P C P B - ~ H O ~ of time per unit time that the ~nsyocardial cell is depolarized, and most likely increases Ga" tinflux per unit time (Edrnan and J6hannsssn 1976). Since @a2' influx per beat in marnnnalian myocardiusn is not likely to increase when the stimulation frequency is increased (Beeler and Reuter 1970). the increase in the force of contraction accompanying an increase in stimu- lation frequency must depend on the cellular accumulation of Ca2+. However. the site of @a2' accumulation under the condi- tion of increased stimulation frequency does not appear to be the sarcsplasmic reticulum, since Sutko cJt csH. (1979) showed that the associated force increase is not inhibited by ryansdiine, an inl-nibitor of sxeoplasmic reticulun-a fanct~on. Also, Lmger et di&. (119'79) have shown that CaZi in superficial sites increases when the beat frequency is increased. The occu~gence of a posltive staircase in frog rnyocardibam (Edraaands er a&. 19&8), which lacks a well-developed sarcoplasrnic reticulu~ra. further indicates that the function sf this organelle is not essential for the force increase aceornpannying an increase in stirnulatiorn frequency. A d a m et ul. ( 1982) reported that arnrinone restores the slow response electromechanicd activity to Kt-depaBarized guinea pig rnyoc~diurn, suggesting that arnrinone, similar to other inhibitors of phosphodiesterase such as the methyl- xanthines (Schneider and Sperelakis B 973, increases the Ca2' influx associated with the slow inward Ca2' ctameamt. Con- versely, halothane inhibits tine Ca2 ' influx associated wiah the slow inward Ga2' current, as Lynch CP Q I . f 198 1 j have shown that it reduces the amplitude and the ~naxiimum rate of rise sf the slow action potential.

As reported by Honge~gger et a&. (1981) for guinea p ~ g papillary muscles. the rested state contractic~n of rabbit papil- lary muscles was enhanced by arnriraeme. Since rested state contractions are most likely dependent on extraceliula Ca" (Lewartawski et al. 1978), the a~bsened reversal by aminone of halothane's negative inotrf-opic effect on the rested state con- traction in contrast with the potentiated state contraction (see beHow) suggests that aminone predo~ninantly counteracts unly one of the actic~ns sf halothane, namely, the anesthetic's effect

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FIG. 4. Effect of aminoa~e and halothane on the developed force generated by single- or paired-pulse stimulation. The frequency of stimulation was 0.2 Hz. The intrapair coupling interval for paired pulses was 300 ms. Mean 4. %EM (PI .= 8) of developed force are shown. S, single pulse; PI , paired pulse, first peak; P2, paired pulse, second peak. Halothane (Hal), 1%. Amrinone (Am), 0.8 rng/nal. *. p < 0.05 cornpad with corresponding control value (paired r-test).

to inhibit the influx of extracellular Ca2 ' . In the presence of aminone, we found that paired-pulse

stimulation produced summation rather than pcsstextrasystcslr9: potentiation. Sutko et szk. (19'79) reported that the contractile response of mammalian rnyocarclilaitar ICP paired-pulse stirnu- lation in the presence of ryanodine, an agent which inhibits sarcoplasmic reticular function, was summationli ke. Frog rnyi~cardium, which I acks a well-developed sarcoplasrnisr retic- ulum is known to show summation rather than postextrasystolic potentiation (Edmands et a&. 1968). Finally, caffeine, an inhibitor of @a" uptake by the sacsplasrnic reticuium, also causes summation instead of postextrasystolic potentiation under the condition of paired-pulse stimulation (Henderson 6%

a / , 1974). Thus, it appears that potentiation depends on a functional sascoplasmic reticulum, and that summation in re- sponse to paired-pulse stimulation occurs when sxcoplasmic reticular function is minima1 or impaired, owing either to the absence sf well-developed organelles (frog), or tc~ the presetace sf an inhibitor such as qanodine or caffeine. If one subtracts the contribution of the late portion of the first peak from the second peak, the forces in response to the first and the second of the pair of stimuli are comparable when sunamation occurs. In other words, in surnrnatiorn, it is v e y likely that each stim- ulus gives ~ s e to almost the same magnitude of force as if the muscles are stimulated by evenly spaced stimuli of twice the frequency. Thus, the sumrnatiosa, even though stimuli are un- evenly spaced, seems to resemble staircase rather than post- extrasystolic potentiation. Hf this interpretation is cc~nect, the enhancement by amriraone of the force of contraction of both

peaks (Pi as well as P2) generated by paired-pulse stimulation does not necessarily mean enhancement by arnrinaone of post- extrasystolic potentiation but more likely a staircaselike effect of arnriwsrae. Ira the presence of anxinone and halothane, the pattern of contraction in response to paired-pulse stimulation was summation, and 0.8 mg/ml of amginone effectively coun- teracted the effect of 1% halothane on the second but not the first peak, suggesting that aminone does not increase Ca" in the intracellular store which is important in potentiated state but increases the influx of extracellular Ca" as in staircase. The pattern of contractile response (summation) to paired-pulse stimulation observed in the presence of aminone suggests that the function of sarcc~plasmic reticulum may be suppressed. This is consistent with the finding of Hongerjiiger eT ok. (1981) that aminone, in high doses. increases relaxation time in a manner similar to theophylline or caffeine. For rat myo- cardium, a tissue in which the csntraetiIe activity is largely determined by the amount of CaZt stored in the sarcoplsrraic reticulum (Fabiato and Fabiato 1977; Stutko and Willerson 1980), amriaone does not cause a positive inotropie effect (Millard et a&. 1980; Azarji and Itiuxtable 1980). This suggests that aminone does not increase Ca2' stored in the ~arcopla~mic reticulum. Hn this regard aminone resembles caffeine, which not only Backs a positive inotrspic effect but actually has a negative inotropic effect in rat myocardium (Hendenon et a/ , 1974).

In conclusion, the results of the present study strongly sug- gest that arnrinone increases the transsarcolernmal influx of Ga2'. whereas it reduces the contribution to contractile activity of Ca2' accumulation, most likely in sarcoplasmic reticulum, under the conditions producing potentiatlsn. This inter- pretation is consistent with the ability c~f aminone to counteract the negative insotropic effect of halothane at Isw but not at high stimulation frequencies and in rested state but not in potentiated state.

This work was supported in part by a National Institute of Health research grant GM 29527 and by a grain-in-aid from the University of Wisconsin, Department of Anesthesiology Research and Development Fund. The authors are grateful to Dr. A, A. Alousi , Sterling-Winthrcsp Research Institute , Rensselaer, PdY for the generous gift of amsinone,

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1386 CAN. B. PHYSHOE. PHARhriACOL. VOL. 62, 1983

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