Gen Pharmac Vol 23, No 6, pp 1135-1138, 1992 Printed m Great Bntam All rights reserved

0306-3623/92 $5 00 + 0 00 Copyright © 1992 Pergamon Press Ltd

E F F E C T S OF M O R P H I N E ON I S O L A T E D R I G H T A T R I A OF T H E RAT M ROMERO,~ M L LAORDEN,2J HERNANDEZ 2. a n d J S SERRANO 1 tDepartment of Pharmacology, Medical School of Sevllla and 2Department of Physiology and Pharmacology, Medical School of Murcla, Spare

(Recewed 26 March 1992) Abstract--I The present study describes the effects of morphine In the absence or presence of naloxone or atropme m the Isolated nght atria of the rat 2 Morphine slgmficantly decreased the auricular chronotroplsm 3 The maximal effect was 10 ___1 0% 4 Slnular results were obtained m reserpnuzed animals (13 ___0 2% maximum) 5 Naloxone (5 × 10-7 or 5 × 10 -6 M) did not change the lnlubltory effects induced by morphine 6 The maximal effect obtamed with morphme m the presence of atropine (5 x 10 -7 M) was 9 + 0 1% slmdar to that obtained with morphine alone 7 These results suggest that oplo~d or vagal mechanisms may not be involved m the cardiac Inhibitory effects mduced by morphine

INTRODUCTION M o r p h i n e has traditionally been t h o u g h t to produce b r a d y c a r d m a n d h y p o t e n s l o n m the adult The d e p r e s s m n o f m y o c a r d m l function by m o r p h i n e has been suggested to be due to a n mteracUon w~th m y o c a r d m l oplold receptors (Rlggs et al, 1986) However, it h a s been d e m o n s t r a t e d m &fferent h e a r t muscle p r e p a r a t i o n s from m a m m a l i a n species, m c l u d m g m a n , t h a t opmtes fad to influence cardmc functaon &rectly, which re&cares t h a t f u n c t m n a l oplold receptors are n o t present postsynaptleally in the h e a r t or, at least, are o f neghglble slgmficance ( N a w r a t h et al, 1989) It ~s possible t h a t e n d o g e n o u s oplolds m a y slgmficantly alter cardmc function by m o d u l a t i o n o f the release of n o r a d r e n a h n e ( M a n t e l h et al, 1987) or acetylchohne ( M u s h a et al, 1989) In this way there is experimental evidence to s u p p o r t the h y p o t h e m t h a t oplold pepUdes m a y function as n e u r o r a n s m m e r s or as n e u r o m o d u l a t o r s m the v a n ous kinds of peripheral effector organs innervated by the sympathetic a n d p a r a s y m p a t h e t m n e u r o n s In order to elucidate the possible m e c h a n i s m s revolved m the cardmc effects o f m o r p h i n e we have mvesUgated the effects o f m o r p h m e o n isolated right atria m the absence a n d presence o f naloxone or a t r o p m e a n d m reserpmmed rats

METHODS

The experiments were carried out on isolated nght atria taken from Sprague-Dawley rats of either sex, weighing 150--200 g The animals were killed by a blow on the head and tmmedlately decapitated The chest was opened w~th a nudsternal incision and the right atria was ~solated by a techmque s~nular to that described previously (Furchgott et al, 1963) The nght atria with smo-astrlal node was suspended In a 10ml organ bath Tyrode solutmn of the *To whom all correspondence should be addressed

following composition (mM) was used NaCI 136 9, KCI 50, Ca 2+ 1 8, C12Mg 1 5, NaH3PO 4 04, NaHCO 3 11 9, Dextrose 0 5 The bathing solution was maintained at 37°C, pH 7 4 and bubbled with 95% 02 and 5% CO 2 The right atna was beating spontaneously and was suspended under a resting tension of 0 5 g and eqmhbrate for 30 nun before the start of the experiments The frequency of spontaneous contracttons was measured by using a force~tsplacement transducer (Grass FT-03) and recorded on a Dynograph Beckman polygraph Morphine solutions were made dady with glass-distilled water and were added to the bath m a volume of 0 1 ml Control concentrations curves to morphine (10-9-10 -4 M) were made m lassues from reserplnlZed and non-reserplmzed rats We also made concentration-response curves for naloxone (10-9-10 -4 M) and atropine (10-t°-10 -4 M, data not shown) ConcentraUon-response curves to morphine were also obtained in the presence of naloxone (5 x 10-7 or 5 x 10-6M) added 5mln before morphine Reserpine (5 mg/kg i p ) was injected 24 hr before the experiments The drug concentration-response curves were generated m a cumulative manner

Drugs The drugs used in this study were morphine hydrochlonde (Alcahber Laboratories), naloxone hydrochlonde (a gift from Merck, Sharp & Dohme), atropine (Sigma) and reserpine (Clba-Gelgy) Analysts of data The effects of morphine alone are expressed as the percent changes from the basehne values Similarly, the effects of morphine in presence of naloxone or atropine are expressed as the percent changes from baseline values after addition of naloxone or atropine Data were analyzed by analysis of variance w~th Student's t-test for individual comparison P < 0 05 was considered slgmficant RESULTS

Forty-three experiments were performed with the above d e s c n b e d t e c h m q u e In all the experiments a s p o n t a n e o u s a n d persistent automatlclty was

1135

M ROMERO et al

1136 105 - -

105

-

95

-

90

-

i

100

95 -

"~

o

m

2

90 0

0

0 85

so

-

q 10

I

I

I

I

I

I

I

9

8

7

6

5

4

3

8s 10

-Log molar concentrahon

I

I

I

I

I

I

8

7

6

5

4

3

-Log molar concentratton

Fig 1 Conccntratmn-response curves for the effect o f morphine on atrial chronotroplsm an non-reserpmlzed ( 0 , n = 7) and tn reserplmzcd rats ( V , n = 8) Each p o i n t

represents the mean 5- SE

obtained The mean control auncular rate before addatlon of any drug was 246 _ 0 1 beats/mm

Effects of morphme Morphine dad not slgmfieantly alter auncular automaUcaty at concentratmns ranging between 1 0 - 9 and 10-7 M However, higher concentrations (10-6-10 -4 M) significantly (P < 0 001) decreased the nomotopm automaUclty The maximal decrease was 1 0 + 0 1% Smular results were obtained m reserpm~zed ammals In this case the maximal effect obtained with morphine was 13 + 0 2 % (Fig 1)

Effects of naloxone The concentrataon-response curve to naloxone on the auncular automatlcaty is shown m Fig 1 Naloxone stgmficantly decreased the nomotoplc automatzoty with all the concentratmns tested,

Fig 3 Effects of morphine on auncular chronotroplsm an absence ( 0 , n = 7) and m presence of naloxone 5 x 10 M (W, n = 7) or 5 x 10 M (V, n = 7) Each point represents the mean 5- SE

10 - g M (10-8-10 - 6 M , P < 0 05, 10 -5 M, P < 0 0 1 , 5 x l 0 -5, 10 - 4 M , P < 0 0 0 1 ) The maximal decrease (28 __. 2 1%) was obtained with the higher concentration tested except

Effects of morphine m presence of naloxone To know whether the effect of m o r p h n e could be attnbuted to actwatmn of a opined receptor, ~t was decided to study the action of morphine m presence o f naloxone (5 x 10 - 7 o r 5 × 10 - 6 M ) The mhihitory effects of morphine on auricular automatlcity was not modified by the presence of naloxone (Fig 3) The max,mal effects obtamed with m o r p h m e in the presence of dafferent concentraaons of naloxone (5 x 10 -7 or 5 x 10 - 6 M ) were 10 + 0 3% and 9 + 0 2 % respectwely These results were mmflar to that obtained with morphme alone (10 + 0 1%)

105 100

I 9

-

-

100

-

95

-

90

-

85

-

A

7

90

G)

v ¢)

2

w

=o 80 -

\

0

0 70

0

0

8O

I

I

I

I

I

I

I

I

10

9

8

7

6

5

4

3

-Log molar concentration Fig 2 Effects of naloxone (0, n = 7) on auricular automatlcaty Each point represents the mean + SE

10

I

I

I

I

I

I

I

9

8

7

6

5

4

3

-Log molar concentration Fig 4 Effects of morphine m absence ( 0 , n = 7) or m presence of atropine 5 x tOM (V, n =7) Each point represents the mean 5- SE

Effects of morphine on rat atria Effects o f morphine m presence o f atropine

Atropine m a concentration (5 x 10 -7 M) &d not alter the effects of morphme on the auricular chronotrop~sm (F~g 4) In th~s case the maximal effect was 9 + 0 1% s~mdar to that described w~th morphine alone (10 +__0 1%)

DISCUSSION Several hnes of evadence have demonstrated that the opmtes have profound effects on the car&ovascular system In this study, m which the effects of opmtes on extracardmc system are ehmmated, morphine caused a s~gmficant decrease on auncular automat~c~ty These results are m agreement w~th other authors those that descnbed cardmc inhibitory effects of morphine (Hegelsen and Refsum, 1987, Laorden et al, 1990) In contrast It has been demonstrated that opmtes d~d not slgmficantly affect the configuration of the mtracellularly recorded action potentml m ventncular heart muscle ~solated from gumea-p~g, rabbit and man (Nawrath et al, 1989) Moreover there is ewdence that morphine reduced tacbycardm m d~fferent expenmental condmons (Felberg and We~, 1986, Given et al, 1986, Zhu and Szeto, 1989) These conflicting results are difficult to interpret because there are many vanables m these stu&es (surgical procedures, &fferent anaesthetic concentrations, autonomic reflexes, species &fferences, &fferent mtnns~c heart rates, etc) On the other hand, naloxone decreased auricular automatlc~ty These results are m agreement w~th prewous stu&es (Huang et al, 1986, Lee and Wong, 1986, Romero et al, 1991) Thus, ~t has recently been reported that naloxone has a &rect electrophys~ological effect m guinea-pig atria and papdlary muscle (Brasch, 1986) Furthermore, ~t has been demonstrated on sheep Purkmje fibers that naloxone has &rect electrophyslologlcal effects which probably underhe ~ts &rect antmrrhythm~c action (Cerba~ et al, 1989) A point of interest m the present results ~s that the inhibitory effects of morphme on auricular automat~clty were not ehmmated when naloxone was present in the organ bath This result suggests that the effects of morphine m the heart are medmted by a non-op~o~d mechamsm of action In contrast the depression of myocardml funcUon by morphme has been suggested to be due to an interaction w~th myocardml op~o~d receptors (Raggs et al, 1986) Furthermore, the electrophys~olog~cal and mechanical effects of opmtes on isolated heart muscle could be due to their interaction w~th myocardial op~o~d receptors (Hegelsen and Refsum, 1987) However, ~t has described that opiates suppress exc~tabflRy of ~solated strips of muscles m a non-stereospec~fic fashion (Same, 1989) On the other hand, ~t has been postulated m the peripheral autonomic nervous system that op~o~d pept~des may play a regulatory role, as neuromodulators, m parasympathetic and sympathetic transnuss~on Few stu&es are avadable for the effects of op~o~d agonlsts m vagal transrmss~on to the heart These stu&es shown that vagal transmlss~on to rabb~t or rat heart can be mh~b~ted by actwat~on of op~o~d

1137

receptors located on the vagus nerve (Koyanagawa et al, 1989, Musha et al, 1989) However, it has been demonstrated that the morphine-produced bradycardm is due to actwat~on of vagal afferents (Wfllette and Sapru, 1982, Kmng and We1, 1983, Ran&ch et al, 1991) In contrast, our results demonstrated that atropine faded to modify the negative chronotroplc effects reduced by morphine These data suggest that the cardiac inhibitory effects reduced by morphine are not medmted by actwatlon of vagal transmission Furthermore, there ~s conslderable expenmental ewdence suggesting that oplold agomsts result in an inhibition of sympathetic transmlss~on m various noradrenerg~cally innervated tissues including heart (Ledda et al, 1985, 1989, ManteU et al, 1987) However, the inhibitory effects reduced by morphine m our preparation were not altered when rats were reserpmlzed These data suggest that the negatwe chronotroplc effects reduced by morphine are not mediated by noradrenerglc mechamsms In conclusion the present results suggest that optold mechanism may not be mvolved m the cardiac inhibitory effects induced by morphine It Is possible that a &rect effect on ~omc currents in cardmc muscle was the mechamsm ~mpllcated In this hne a recent study (Same et al, 1991) has demonstrated that the antmrrhythm~c effects of oplo~d drugs are not medmted by op~o~d receptors suggesting that the op~old agomsts as well as oplo~d antagomst interact w~th voltage-dependent sodmm channels This nonop~o~d action of opmtes should be taken into cons~deratlon when interpreting the pharmacological effects of oplo~d drugs REFERENCES Brasch H (1986) Influence of the optical isomers (+) and ( - ) naloxone on beating frequency, contractile force and action potentials of guinea-pig isolated car&ac preparations Br J Pharmac 88, 733-740 Cerba~ E, Cavalcabo P B, Masm~ I, Porclattl F and Mugelh A (1989) Anttarrhytmlc properties of naloxone an electrophyslolog~calstudy on sheep cardmc Purkmge fibers Fur J Pharmac 162, 491-500 Felberg W and Wel E (1986) Analysis of car&ovascular effects of morphine m the cat Neurosczence 17, 495-506 Furchgott R F, Klerpekar B M and Rleker M (1963) Actmns and interaction of norepmephnne, tyramme and cocaine on aorUc strip of rabb~t and left atria of guinea-pig and cat J Pharmac exp Ther 142, 39-42 Gwen M B, Samder G E and Gtles T D (1986) Non opiate and penpheral opiate car&o-vascular effects of morphine m conscious-dogs Life Sct 38, 1299-1303 Hegelsen K G and Refsum H (1987) Arrhytmogemc, antarrhythmlc and motroplc properties of oplolds Pharmacology 35, 121-139 Huang X D, Lee A Y S, Wong T M, Zhan C Y and Zhao Y Y (1986) Naloxone inhibits arrhythmms reduced by coronary artery occlusion and reperfuslon m anaesthetized dogs Br J Pharmac 87, 475-477 Klang J G and Wel E T (1983) InhlbRlon of an oploldevoked vagal reflex m rats by naloxone, SMS 201-995 and ICI 154,129 Reg Pepttdes 6, 255-262 Koyanagawa H, Musha T, Kamda A, Klmura T and Satoh S (1989) Inhlbmon of vaghal transmission by cardmc sympathetic nerve stlrnulat~onm the dog possible involvement of oplold receptor J Pharmac exp Ther 250, 1092-1096

1138

M ROMEROet al

Laorden M L , Hernandez J , Carceles M D , Mlralles F S and Ping M M (1990) Interactmn between halothane and morphine on ~solated heart muscle Eur J Pharmac 175, 285-290 Ledda F , Mantelh L and Cort~ V (1985) Sensmwty to dynorphm-(1-13) of the presynaptlc inhibitory opmte receptors of the guinea-pig heart Eur J Pharmac 117, 377-380 Ledda F , Cort~ V and Mantelh L (1989) Indirect ewdence for a role of prostaglandms as second messengers of the prejunctlonal effect of oplolds m guinea-pig ventncular preparahons Eur J Pharmac 162, 323-327 Lee A Y S and Wong T M (1986) Naloxone attenuates augmentation of cAMP levels and arrhythlmas following myocardml lschaemm and perfuslon m the isolated perfused rat heart Chn exp Pharmac Physwl 13, 707-710 Mantelh L , Cortl V and Ledda F (1987) On the presence of oplotd receptors m the guinea-pig ventncular tassue Gen Pharmac 18, 309-311 Musha T , Satoh E , Koyanagawa H , K~mura T and Satoh S (1989) Effects of oplold agonlsts on sympathetic and parasympathetic transmission to the dog heart J Pharmac exp Ther 250, 1087-1091 Nawrath H , Rupp J , Jakob H , Sack U , Mertzlufft F and Dick W (1989) Fadure of oplolds to affect excRatlon

and contracuon m ~solated ventncular heart muscle Exper,entla 45, 337-339 Randlch A , Thurston C L , Ludwmg P S, Tlmmerman M R and Gebhart G F (1991) Actmoceptton and cardiovascular responses produced by mtravenous morphine the role of vagal afferents Brain Res 543, 256-270 Paggs T R , Yano Y and Varglsh T (1986) Morphine depression of myocardial function Czrc Shock 19, 31-38 Romero M , Laorden M L , Hernandez J and Serrano J S (1991) Evidence for involvement of catecholammes m the effect of morphine on ventncular automataoty m the rat J Auton Pharmac I1, 97-103 Same Y (1989) Non opiate effects of oplold agomsts and antagomsts on cardiac muscle Adv Bzosc~ 75, 551-554

Same Y , Fllstem A , and Oppenheimer E (1991) Antlarrhythmlc actlvRles of op~old agomsts and antagomsts and their stereolsomers Br J Pharmac 102, 696-698 Wfllette R N and Sapru H N (1982) Peripheral versus central card~oresplratory effects of morphine Neuropharmacology 21, 1019-1026 Zhu Y S and Szeto H (1989) Morplune-mduced tachycard m m fetal lambs a bell shaped dose-response curve J Pharmac exp Ther 249, 78-82