- Email: [email protected]
The role of calcium in supersensitivity to the inotropic effects of norepinephrine
European Journal of Pharmacology, 50 (1978) 359--367
359
© Elsevier/North-Holland Biomedical Press
T H E R O L E O F C A L C I U M IN S U P E R S E N S I T I V I T Y T O T H E I N O T R O P I C E F F E C T S O F NOREPINEPHRINE THOMAS E. TENNER, Jr., JOHN H. McNEILL and OLIVER CARRIER, Jr. Department of Pharmacology, Texas Tech University School of Medicine, P.O. Box 4569, Lubbock, Texas 79409, U.S.A.
Received 13 January 1978, revised MS received 25 April 1978, accepted 3 May 1978
T.E. TENNER, Jr., J.H. McNEILL and O. CARRIER, Jr., The role of calcium in supersensitivity to the inotropic effects of norepinephrine, European J. Pharmacol. 50 (1978) 359--367. Experiments using electrically stimulated rabbit left atria have demonstrated that supersensitivity to the inotropic effects of norepinephrine can be induced by either chronic reserpine pretreatment or hypothermia (lowering the temperature of the bathing medium). These two experimental conditions for inducing supersensitivity were not additive implying that they shared a common mechanism of action. Norepinephrine had no significant effect on the amplitude of a potentiated contraction of the rabbit atrium when the temperature was reduced from 37 to 30°C or following pretreatment with reserpine (30 or 37°C). Under these same conditions the EDs0 of norepinephrine on the normal contraction was reduced. It is concluded that both reserpine pretreatment and hypothermia induce supersensitivity to the inotropic effects of norepinephrine by enhancing the cellular store of activator calcium while not affecting the ability of norepinephrine to release activator calcium. Hypothermia- and reserpine-induced supersenstitivity Potentiated contraction
Norepinephrine
1. I n t r o d u c t i o n
p e r f o r m e d . While t h e r e are several theories c o n c e r n i n g h y p o t h e r m i a - i n d u c e d supersensitivity in response t o s y m p a t h o m i m e t i c drugs, t h e p h e n o m e n o n p r o b a b l y occurs in t h e series o f events c o n s e q u e n t to t h e stimulation o f a d r e n o c e p t o r s b u t n o t in the r e c e p t o r binding site itself ( R e i n h a r d t et al., 1972; E n d o h et al., 1975). C o n s i s t e n t with this h y p o t h e s i s , Reiter and Stickel ( 1 9 7 0 ) have d e m o n s t r a t e d t h a t t h e d o s e - - r e s p o n s e curves for b o t h symp a t h o m i m e t i c agents and calcium are shifted to t h e right as t e m p e r a t u r e is raised. It is possible t h a t an altered utilization o f calcium b y m y o c a r d i a l cells m a y be the e v e n t responsible for t h e a p p a r e n t change in affinity o f s y m p a t h o m i m e t i c amines as t e m p e r a t u r e is lowered. Chronic p r e t r e a t m e n t with reserpine, an alkaloid k n o w n t o d e p l e t e c a t e c h o l a m i n e f r o m the heart, can i n d u c e supersensitivity t o t h e c h r o n o t r o p i c effects o f b o t h calcium and
Supersensitivity has b e e n d e f i n e d as t h e p h e n o m e n o n i n which t h e a m o u n t o f a substance r e q u i r e d t o p r o d u c e a given biological response is less t h a n n o r m a l ( F l e m i n g et al., 1 9 7 3 ) . This p h e n o m e n o n is n o r m a l l y i n d u c e d b y e i t h e r surgical or p h a r m a c o l o g i c a l denervat i o n o f the e f f e c t o r tissue and is t h o u g h t to reflect a c o m p e n s a t o r y change in the postj u n c t i o n a l e f f e c t o r cell in response to the subsequent, c h r o n i c loss o f n e u r o t r a n s m i t t e r (Fleming, 1 9 7 6 ) . R e c e n t l y , h o w e v e r , supersensitivity to the c h r o n o t r o p i c ( T r e n d e l e n b u r g , 1 9 6 8 ; Opperm a n n et al., 1 9 7 2 ; WSppel and T r e n d e l e n burg, 1 9 7 3 ) and i n o t r o p i c ( R e i n h a r d t et al., 1 9 7 2 ; B r o a d l e y , 1974) effects o f s y m p a t h o m i m e t i c agents has been achieved in m y o cardial p r e p a r a t i o n s b y m e r e l y lowering the t e m p e r a t u r e at which an e x p e r i m e n t is to be
Calcium
360
catecholamines (Fleming, 1976). In contrast, pretreatment with reserpine was thought not to induce supersensitivity to the inotropic effects of catecholamines and calcium (Taylor et al., 1974). Broadley and Lumley (1977) have reported, however, that reserpine pretreatment does induce a selective or specific supersensitivity to the inotropic effects of isoproterenol and salbutamol. As Broadley and Lumley were unable to demonstrate supersensitivity to the inotropic effects of either calcium or histamine, they concluded that this "selective" supersensitivity might be due to either an increased efficacy or proliferation of the fi-receptor. More recently, Duncan and Broadley (1977) have demonstrated that the selective supersensitivity induced by reserpine pretreatment to the inotropic effects of isoproterenol was masked if the temperature was lowered from 38 to 30 or 25°C. These authors speculated that both reserpine-induced and hypothermiainduced supersensitivity might result due either to an increase in the fi-receptor population or the relationship between receptor occupation and the response. However, Tenner and Carrier (1978) have reported that supersensitivity to the inotropic effects of calcium can be demonstrated in the electrically driven rabbit atria following reserpine pretreatment. This supersensitivity could be demonstrated at both 37 and 30°C without an apparent masking effect of the hypothermia-induced supersensitivity. As calcium may play a crucial role in both reserpine-induced and hypothermia-induced supersensitivity phenomena, it was decided that a study of these two forms of supersensitivity to the inotropic effects of norepinephrine and calcium might prove fruitful. One means of determining the utilization of calcium by cardiac tissue is by studying "potentiated" contractions on the first beat following a period of quiescence (Wood et al., 1969). Langer et al. (1975) noted that the potentiated contraction was a measure of the degree of activation of the myocardial cell and, therefore, a measure of the relative
T.E. TENNER, JR. ET AL.
amount of calcium released to the myofilaments. It was decided therefore to compare potentiated contractions obtained with left atria from untreated and reserpine-pretreated rabbits following various concentrations of norepinephrine and calcium at various temperatures in the hope of further illustrating the role of calcium in supersensitivity phenomena.
2. Materials and methods
2.1. Tissue preparation and procedures New Zealand white rabbits of either sex weighing between 1.0 and 1.5 kg were either chronically pretreated (7 days) with daily intramuscular injections of 0.1 mg/kg reserpine (Serpasil-CIBA) or were used as untreated controls. 24 h after the last pretreatment, the rabbits were stunned by a blow on the head and exsanguinated. After thoracot o m y , the hearts were excised, and immediately placed in oxygenated Chenoweth-Koelle solution (CKS) of the following composition (in mM): NaC1 120; KC1 5.63; CaC12 2.0; dextrose 9.7; MgC12 2.0; NaHCO3 25.0. Following separation from the ventricles, the left atrium was m o u n t e d vertically in a tissue chamber containing 5 0 m l of CKS which was continuously oxygenated with a gas mixture of 95% 02--5% CO2. The pH of the solution was maintained at 7.3 + 0.05 and the temperature was held at either 30 or 37°C depending upon the experiment to be performed. One portion of the atrium was secured to a Plexiglas electrode apparatus modified from that developed by Blinks (1966). The other portion was connected via a thin chain to a Grass FT-03 strain gauge transducer. The atrium was allowed to equilibrate for 45 min under a mechanically applied diastolic tension of 2 g. During equilibration, the bathing solution was completely changed every 15 min. The left atrial preparations were quiescent unless excited electrically b y the electrodes.
CALCIUM AND CARDIAC SUPERSENSITIVITY Two platinum wires were situated such that the Plexiglas clamp secured the lower portion of the atrial tissue against the heads of the electrodes. Preparations which beat spontaneously were discarded. Electrical stimulation was applied by a Grass SD-5 stimulator with a duration of 3 msec at a voltage approximately twice the threshold voltage (1--2 V). Preliminary studies using propranolol and atria from animals treated 24 h previously with reserpine indicated that this intensity did not result in significant release of endogenous neurotransmitter. The frequency of stimulation during the equilibration period and the subsequent experiments was 1.6 Hz. Isometric tension was recorded on a Grass Model 7 polygraph. For the purposes of this study, isometrically developed contractile force will be referred to as developed force. The developed force recorded at the end of the equilibration period will be referred to as the Basal Developed Force (BDF).
2.2. Experimental design Dose--response relationship for norepinephrine (L-arterenol, Sigma) were obtained using atria from reserpine-pretreated rabbits and untreated controls at 30 and 37°C. Following the addition of a given dose of norepinephfine, the atria were allowed to achieve a new steady state developed force. Upon achieving the plateau force, the stimulator was turned off and a 30 sec period of quiescence ensued. Upon resumption of stimulation, the first beat following the quiescent period, i.e. the potentiated contraction was recorded. A period of 30 sec was chosen as the amplitude of the potentiated contraction was found to be maximal following this quiescent period for atria tested at both 30 and 37°C. This observation is in agreement with that of Spikler and Cervoni (1969). Upon reattaining the steady state developed force achieved prior to the quiescent period, a higher dose of norepinephrine was added. This process continued until the maximal developed force (MDF) and maximal amplitude of the po-
361 tentiated contraction were achieved. Only one dose--response relationship was obtained for each atria. Potentiated contractions were also obtained for atria at three different calcium concentrations (0.6, 2.0 and 8.0 raM). After equilibration in normal CKS, the bathing medium was changed to a CKS containing the desired calcium concentration. The atria were allowed to achieve a new steady state developed force prior to obtaining potentiated contractions. These responses were obtained in a similar manner as those for norepinephrine.
2.3. Analysis and statistics The presence of supersensitivity was determined by comparison of geometric mean EDs0 values (Fleming et al., 1973; Fleming et al., 1972). The EDs0 values for norepinephrine were obtained graphically from the individual dose--response curves following conversion of the absolute response at each concentration to percentages of maximum response. The EDs0 values were then compiled to yield the geometric mean -+ 95% confidence interval. In addition, estimates of the change in sensitivity to norepinephrine are described as the ratio of the geometric mean EDs0 values. All other data are absolute values presented as arithmetic means-+ S.E.M. Statistical significance was determined by use of the Student's t-test. Differences were considered to be significant if P < 0.05.
3. Results Fig. 1 illustrates the influence of hypothermia (decreasing the temperature of the bathing medium from 37 to 30°C) on norepinephrine-induced increases in developed force in left atria from untreated rabbits. At subthreshold doses of norepinephrine (10 -9 M), a hypothermia-induced increase in basal developed force (BDF) was apparent. In contrast, the maximum developed force (MDF) ohtained to high doses of norepinephrine was
IEDs° i
362 6.0 __
5.0
v
4.0
30.0
T.E. TENNER, JR. ET AL. 7.0 60 50
~°°I
O
30
22.0
40
14.0
30
ED50
o >
X
20
,
C~
~
1
I 9
i 8
I 7
i 6
z 5
i 4
30oC
37oc
9
Fig. 1. The influence of temperature on the dose-response relationship and Geometric Mean EDs0 value for norepinephrine. Open circles represent responses obtained at 30°C while the open triangles represent responses obtained at 37°C. Each point represents the mean ± S.E.M. of 6--8 experiments. Vertical bars on the EDs0 values represent the 95% confidence interval for the Geometric Means.
not significantly altered by the temperature at which the experiment was performed. When the EDs0 values for norepinephrine were calculated for the two temperatures, bY" pothermia-induced supersensitivity was apparent. The EDs0 value for norepinephrine at 30°C was approximately one third of that at 37°C indicating a threefold increase in sen-
5.0
±
.oofi ED50
4.0 ,2°
~,
3.0 -$8
x
20
22.0 14.0
Z 1.0
6.0 I
I
I
9
8
7
-Log ENE]
10 J
-Log [NE] (M}
>
×
2.0
6.0
(~1 I
I
I
6
5
4
r~ 37%
37~C +RES.
Fig. 2. The influence of reserpine pretreatment on the dose-response relationship and Geometric Mean EDs0 value for norepinephrine when tested at 37°C. Open triangles represent untreated controls while closed triangles represent atria from reserpine-pretreated rabbits. Each point represents the mean ± S.E.M. of 6--8 experiments. Vertical bars on the EDs0 values represent the 95% confidence interval for the Geometric Means.
22.0
o
I 8
I 7
I 6
I 5
I 4
°°nh 14.0
30oC
30cC +RES.
- Lo9 [NE] (MI
Fig. 3. The influence of reserpine pretreatment on the dose-response relationship and Geometric Mean EDso value for norepinephrine when tested at 30°C. Open circles represent untreated controls while closed circles represent atria from reserpine-pretreated rabbits. Each point represents the mean ± S.E.M. o f 8-10 experiments. Vertical bars on the EDs0 values represent the 95% confidence interval for the Geometric Means.
sitivity at the lower temperature. Fig. 2 illustrates the influence of reserpine pretreatment upon norepinephrine-induced increases in developed force when the experiments were performed at 37°C. In this group of experiments, neither the BDF or the MDF of atria from reserpine-pretreated rabbits were significantly greater than those from untreated animals. While the BDF was increased, significance was not obtained in contrast to a previous report by Tenner and Carrier (1978). When the EDs0 values obtained at 37°C were compared, the value for atria from reserpine-pretreated animals was approximately one third of that from untreated controls. This would indicate a reserpine-induced supersensitivity as the sensitivity of atria from pretreated animals was threefold greater than untreated controls. It is interesting to note that the EDs0 values obtained for atria from untreated rabbits at 30°C and those for atria from reserpine-pretreated rabbits at 37°C were not significantly different from each other (figs. 1 and 2). Fig. 3 illustrates the effects of reserpine pretreatment upon norepinephrine-induced
CALCIUM AND CARDIAC SUPERSENSITIVITY increases in developed force when the experiments were performed at 30°C. In this group of experiments, both the BDF and MDF were greater in atria from reserpine-pretreated rabbits than from untreated controls. In contrast, however, the EDs0 values for atria of untreated and reserpine-pretreated animals obtained at 30°C were not significantly different. It is apparent that hypothermiainduced supersensitivity and reserpine-induced supersensitivity were not additive, i.e. the EDs0 values for atria from reserpine-pretreated rabbits were not significantly different when obtained at 37 and 30°C (figs. 2 and 3). As stated earlier, Tenner and Carrier (1978) reported that reserpine-induced supersensitivity to calcium was not masked by hypothermia-induced supersensitivity. It was therefore concluded that the norepinephrine-induced increases in developed force might be dependent upon the utilization of calcium as influenced by either hypothermia or reserpine pretreatment. It was of interest then to examine calcium utilization by the atrial preparations under the various experimental conditions. The potentiated contraction (the first beat obtained following a thirty second period of quiescence) was chosen as a measure of the relative a m o u n t of calcium released to the myofilaments (Langer et al., 1975). It was first necessary to demonstrate the dependence o f the potentiated contraction amplitude on the extracellular calcium concentration. Figure 4 illustrates this relationship. The amplitude of the potentiated contraction also depends upon the temperature and whether or not the atria used are from reserpine-pretreated or untreated rabbits. As is obvious from fig. 4, the potentiated contraction amplitude obtained with 2.0 mM calcium at 37°C < 37°C + reserpine pretreatment < 30°C < 30°C + reserpine pretreatment. In other words, atria from reserpine-pretreated animals produced amplitudes greater than untreated controls at each of the temperatures studied. Fig. 5 illustrates the influence of temperature on the amplitude of the potentiated contraction obtained using various doses of
363
8.0
7,0 -~
i
6.0
tJ
4.0
"5
3.0
~
2.0 1.0
30°C +RES,
30oC
37°C ÷RES
37oC
Fig. 4. The influence of calcium on the amplitude of the potentiated contraction obtained with atria from either reserpine-pretreated or untreated rabbits at both 30 and 37°C. (~) 0.2 mM, (..) 2.0 mM, (~) 8.0 mM CaC12. Vertical bars represent the S.E.M. of 11-14 experiments at each calcium concentration.
norepinephrine. Note that at subthreshold doses of norepinephrine, the hypothermiainduced increase in the potentiated contraction amplitude, as evidenced, in fig. 4, was
8.0 7.0 6.0
I '5
5.o
i ~
4.0
~
30
6
20 1.0
9
8
7
6
5
4
-Log [NE] (M)
Fig. 5. The influence of temperature on the amplitude of the potentiated contraction obtained for various doses of norepinephrine using atria from untreated rabbits. (~) represents atria tested at 30°C while (•) represents atria tested at 37°C. Vertical bars represent the S.E.M. of 6--8 experiments.
364
T.E. TENNER, JR. ET AL. 6.0
8.0
50
70
i iili
4o U~
3.0
5
20
g ~_
1.0
9
8
7
6
5
6.0
5.0 4.0 30
4
- L o g [NE] {M)
Fig. 6. The influence of reserpine on the amplitude of the potentiated contraction obtained for various doses of norepinephrine at 37°C. (~) represents atria from reserpine-pretreated rabbits while (a) represents atria from untreated rabbits. Vertical bars represent the S.E.M. of 6--8 experiments.
apparent. While norepinephrine increased the amplitude of the potentiated contraction when studied at 37°C, it had no significant effect on the amplitude of potentiated contractions obtained at 30°C. It is important to remember that norepinephrine increased developed force at both temperatures studied (fig. 1). When studied at 37 ° C, atria from reserpinepretreated rabbits produced potentiated contractions of significantly greater amplitude than controls (fig. 6). In a similar fashion as observed for atria studied at 30°C, norepinephrine did not further increase the amplitude of the potentiated contractions of atria from reserpine-pretreated animals, while increasing that for atria from control animals. At a temperature of 30°C, atria from reserpine-pretreated rabbits developed potentiated contractions of significantly greater amplitude than controls (fig. 7). Norepinephrine did not further increase the amplitude of the potentiated contractions of atria from either control or reserpine-pretreated rabbits under the hypothermic condition. There would appear to be a correlation between the EDs0 values reported in figs. 1, 2, and 3 and the ability of norepinephrine to alter the amplitude of the potentiated con-
o-
2.0
H
1.0
i 9
V//A I 8
7
6
5
-Log ~NE] ',M)
Fig. 7. The influence of reserpine on the amplitude of the potentiated contraction obtained for various doses of norepinephrine at 30°C. ([~) represents atria from reserpine-pretreated rabbits while ([]) represents atria from untreated rabbits. Vertical bars represent the S.E.M. of 8--10 experiments.
traction. Reserpine pretreatment and hypothermia resulted in a decrease in the ED~0 values obtained for norepinephrine thus indicating supersensitivity. These two experimental conditions also produced an increase in the potentiated contraction such that no further increase was produced by norepinephrine.
4. Discussion The present study has demonstrated that both chronic reserpine pretreatment and hypothermia will induce supersensitivity, as determined by geometric mean EDs0 values, to the inotropic effects of norepinephrine. The supersensitivity was associated with those experimental conditions where the amplitude of the potentiated contraction could not be further enhanced by norepinephrine. Recently, Duncan and Broadley (1977) published results similar to those described in the present paper. These authors reported that hypothermia could mask the supersensitivity induced by reserpine pretreatment to the inotropic effects of isoproterenol and sal-
CALCIUM AND CARDIAC SUPERSENSITIVITY butamol. In an earlier study, Broadley and Lumley {1977) proposed that the supersensitivity induced by chronic reserpine pretreatment was selective for fi-agonists as they could not demonstrate supersensitivity to either calcium or histamine. It was suggested that the mechanism of action for this selective supersensitivity may result from either an increase in the fi-receptor population or a change in the relationship between receptor occupation and response. Duncan and Broadley (1977) suggested that these two explanations might also apply to the mechanism of hypothermiainduced supersensitivity as it had the ability to mask the supersensitivity induced by reserpine pretreatment. In contrast, Tenner and Carrier (1978) reported that supersensitivity to the inotropic effects of calcium could be induced by chronic reserpine pretreatment and by hypothermia. In addition, reserpine-induced supersensitivity to the inotropic effect of calcium was not masked by that induced by hypothermia. It would appear, therefore, that these two experimental conditions (reserpine pretreatment and hypothermia) induce supersensitivity to the inotropic effects of calcium through different mechanisms as their effects on sensitivity are aditive i.e. neither masks the effects of the other (Tenner and Carrier, 1978). On the other hand, based on the data of the present study as well as that of Duncan and Broadley (1977), reserpine pretreatment and hypothermia appear to induce supersensitivity to the inotropic effects of norepinephrine via the same mechanism i.e. hypothermia masks the effects of reserpine pretreatment and vice versa. One explanation for these results could be that reserpine pretreatment and hypothermia induce supersensitivity to the inotropic effects of calcium via different alterations of calcium utilization (Tenner and Carrier, 1978) while the supersensitivity to norepinephrine results from some common change produced in the fl-receptor (Broadley and Lumley, 1977; Duncan and Broadley, 1977). Another possibility is that reserpine
365 pretreatment and hypothermia influence different steps in the sequence of events resulting in excitation--contraction coupling, and that the inotropic effect of norepinephrine is enhanced in the same manner by both influences. In order to test the latter hypothesis an index of calcium utilization of cardiac muscle was required. Langer et al. (1975)stated that the magnitude of the first beat following a period of quiescence was a measure of the degree of activation of the muscle and therefore of the relative amount of calcium released to the myofilaments. In the present study the amplitude of the potentiated contraction was quantitated and used as an index of calcium utilization. Wood et al. (1969) concluded that the amplitude of the potentiated contraction dependend upon the presystolic level of intracellular calcium bound to specific (rapid release) sites and that this amount of activator calcium was predetermined by the frequency of stimulation, the temperature, the extracellular calcium concentration, and the elapsed time since termination of the last absolute refractory period (i.e. the period of quiescence). Wood et al. postulated that with each depolarization, "the amount of calcium ions dissociated from rapid release sites in the sarcoplasmic reticulum and other intracellular membrane surfaces.., is a relatively constant fraction of the total quantity of calcium stored at these release sites. The greater the amount of calcium present in these structures, the greater the amount dissociated to intracellular free calcium ions by a given action potential". As indicated by increases in the amplitude of potentiated contractions obtained in the present study, reserpine pretreatment and hypothermia both appear to increase the functional amount of activator calcium stored for future release to the myofilaments. Reserpine pretreatment probably increases this store through a different mechanism than hypothermia as an "additive" effect upon the amplitude of the potentiated contraction was observed at each temperature studied. This
366
hypothesis is consistent with the data of Tenner and Carrier (1978) in that reserpineinduced supersensitivity to calcium could be demonstrated under hypothermic conditions. According to Wood et al. (1969), a certain fixed fraction of this store of activator calcium is released for the development of force with each depolarization. As this store is increased b y either reserpine pretreatment, hypothermia, or both, the amount of activator calcium released with each depolarization is increased even though the fraction released remains fixed. This could account for the increases in BDF achieved with hypothermic conditions and following chronic reserpine pretreatment. It should be noted that while atria from reserpine pretreated animals tested at 37°C displayed a greater BDF than untreated controls, the difference was not significant. This finding is interpreted as an artifact of the given experiments as a significant increase in BDF was observed for reserpine pretreated atria at 37°C in other experiments testing calcium (Tenner and Carrier, 1978). The ability of norepinephrine to produce a positivie inotropic effect lies in several postulated mechanisms. Norepinephrine is thought to increase transmembrane calcium influx (Rasmussen et al., 1972; Meinertz et al., 1973), increase sequestration of calcium (Katz and Repke, 1973) and increase the release of stored activator calcium. (Sinebourne and White, 1970; Fabiato and Fabiato, 1975). Increasing calcium influx and sequestration would result in increasing the size of the stored calcium pool. If reserpine pretreatmerit or hypothermia increased the amount of activator calcium available for release, the importance of these two latter mechanisms would be diminished. On the other hand, each dose of norepinephrine would increase the a m o u n t of stored calcium released to a greater extent than in untreated atria at 37°C. At 37°C, each dose of norepinephrine would have to increase the size of the activator calcium pool by increasing calcium influx and sequestration as well as increase the release of this growing calcium pool. This hypothesis
T.E. TENNER, JR. ET AL.
is consistant with the results of the present study. Norepinphrine was able to increase the amplitude of the potentiated contraction only in untreated atria at 37 ° C. On the other hand, no significant increase in the amplitude of the potentiated contraction was observed with norepinephrine in atria from either reserpine pretreated animals or in untreated atria studied at 30°C. Because reserpine pretreatment and hypothermia increased the amount of calcium available for release, each dose of norepinephrine could release more calcium than was possible for the same dose in untreated atria at 37°C. This would explain why the EDs0 values for reserpine pretreated atria at 37 and 30°C were the same as that for untreated atria at 30°C as well as w h y these EDs0 values were significantly less than that for untreated atria at 37 ° C. It would appear therefore that the mechanism by which reserpine pretreatment and hypothermia induce supersensitivity could be by increasing the functional amount of activator calcium which can be released to the myofilaments and not by altering the ability of norepinephrine to release that calcium. This conclusion is consistant with the observation that supersensitivity to the inotropic effect of calcium is induced by reserpine pretreatment and hypothermia in an additive fashion (Tenner and Carrier, 1978).
Acknowledgements This work was supported in part by the National Institutes of Health (NIH) Grant No. HL17899, AFOSR Grant No. 76-3029, and the British Columbia Heart Foundation.
References Blinks, J.R., 1966, Field stimulation as a means of effecting the graded release of autonomic transmitter in isolated heart muscle, J. Pharmacol. Exptl. Therap. 151,221. Broadley, K.J., 1974, The interpretation of doseresponse curves expressed by various graphical means to determine the effects of temperature
CALCIUM AND CARDIAC SUPERSENSITIVITY on the resonses of guinea-pig isolated atria to isoprenaline, Arch. Intern. Pharmacodyn. 210,147. Broadley, K.J. and P. Lumley, 1977, Selective reserpine-induced supersensitivity of the positive inotropic and chronotrpic responses to isoprenaline and slabutam01 in guinea-pig isolated atria, Brit. J. Pharmacol. 59, 51. Duncan, C. and K.J. Broadley, 1977, The influence of temperature upon reserpine-induced supersensitivity of guinea-pig isolated atria to isoprenaline and salbutamol, Naunyn-Schmiedeb. Arch. Pharmacol. 297,163. Endoh, M., J. Wagner and H.J. Schiimann, 1975, Influence of temperature on the positive inotropic effects mediated by ~-,/3-adrenoceptors in the isolated rabbit papillary muscle, Naunyn-Schmiedeb. Arch. Pharmacol. 287, 61. Fabiato, A. and F. Fabiato, 1975, Relaxing and inotropic effects of cyclic AMP on skinned cardiac cells, Nature 253,556. Fleming, W.W., 1976, Variable sensitivity of excitable cells: Possible mechanisms and biological significance, Rev. Neurosci. 2, 43. Fleming, W.W., J.J. McPhillips and D.P. Westfall, 1973, Postjunctional supersensitivity and subsensitivity of excitable tissues to drugs, Ergeb. Physiol. 68, 55. Fleming, W.W., D.P. Westfall, I.S. De La Lande and L.B. Jehlett, 1972, Log-normal distribution of equieffective doses of norepinephrine and acetylcholine in several tissues, J. Pharmacol. Exptl. Therap. 1 8 1 , 3 3 9 . Katz, A.M. and K.I. Repke, 1973, Calcium membrane interactions in the myocardium: Effects of ouabain, epinephrine, and 3',5'-cyclic adenosine monophosphate, Amer. J. Cardiol. 31,193. Langer, G.A., S.D. Serena and L.M. Nudd, 1975, Localization of contractile dependent calcium: comparison of manganese and verapamil in cardiac and skeletal muscle, Amer. J. Physiol. 229, 1003. Meinertz, T., H. Naurath and H. Scholz, 1973, Stimulatory effects of DB-c-AMP and adrenaline on myocardial contraction and ~SCa exchange. Experiments at reduced concentration and low frequencies of stimulation, Arch. Pharmacol. 279, 327. Oppermann, J.A., C.F. Ryan and C.O. Haavik, 1972,
367 The role of metabolism in temperature-dependent supersensitivity of guinea-pig atria to sympathomimetic amines, European J. Pharmacol. 18,266. Rasmussen, H., D.B.P. Goodman and A. Tenenhouse, 1972, The role of cyclic AMP and calcium in cell activation, Crit. Rev. Biochem. 1, 95. Reinhardt, D., J. Wagner and H.J. Schiimann, 1972, Influence of temperature on the sensitivity of the /3-receptors and the contractility of guinea-pig atrium, Naunyn Schmiedeb. Arch. Pharmacol. 275, 95. Reiter, M. and F.J. Stickel, 1970, Zur Frage einer therapeutischen Steigerung der Kontraktilit/it bei Abkfihlung des Herzens, Klin. Wschr. 4 8 , 9 3 5 . Shinebourne, E. and R. White, 1970, Cyclic AMP and calcium uptake of the sarcoplasmic reticulum in relation to increased rate of relaxation under the influence of catecholamines, Cardiovasc. Res. 4, 194. Spikler, B. and P. Cervoni, 1969, Electrical membrane activity and frequency-force relations in normal, denervated and reserpine pretreated cat heart muscle, J. Pharmacol. Exptl. Therap. 168, 60. Taylor, D.A., D.P. Westfall and W.W. Fleming, 1974, The effect of reserpine treatment on the chronotropic and inotropic sensitivities of the perfused guinea-pig heart to norepinephrine and calcium, European J. Pharmacol. 29, 102. Tenner, T.E. and O. Carrier, 1978, Reserpine-induced supersensitivity to the chronotropic and iontropic effects of calcium in rabbit atria, J. Pharmacol. Exptl. Therap. 205,183. Trendelenburg, U., 1968, The effect of cocaine on the pacemaker of isolated guinea-pig atria, J. Pharmacol. Exptl. Therap. 161,222. Wood, E.H., R.L. Heppner and S. Weidmann, 1969, Inotropic effects of electric currents. I. Positive and negative effects of constant electric currents or current pulses applied during cardiac action potentials. II. Hypothesis: calcium movements, excitation-contraction coupling and inotropic effects, Circulation Res. 24,409. WSppel, W. and U. Trendelenburg, 1973, Temperature-dependent supersensitivity of isolated atria to catecholamines, European J. Pharmacol. 23,302.
359
© Elsevier/North-Holland Biomedical Press
T H E R O L E O F C A L C I U M IN S U P E R S E N S I T I V I T Y T O T H E I N O T R O P I C E F F E C T S O F NOREPINEPHRINE THOMAS E. TENNER, Jr., JOHN H. McNEILL and OLIVER CARRIER, Jr. Department of Pharmacology, Texas Tech University School of Medicine, P.O. Box 4569, Lubbock, Texas 79409, U.S.A.
Received 13 January 1978, revised MS received 25 April 1978, accepted 3 May 1978
T.E. TENNER, Jr., J.H. McNEILL and O. CARRIER, Jr., The role of calcium in supersensitivity to the inotropic effects of norepinephrine, European J. Pharmacol. 50 (1978) 359--367. Experiments using electrically stimulated rabbit left atria have demonstrated that supersensitivity to the inotropic effects of norepinephrine can be induced by either chronic reserpine pretreatment or hypothermia (lowering the temperature of the bathing medium). These two experimental conditions for inducing supersensitivity were not additive implying that they shared a common mechanism of action. Norepinephrine had no significant effect on the amplitude of a potentiated contraction of the rabbit atrium when the temperature was reduced from 37 to 30°C or following pretreatment with reserpine (30 or 37°C). Under these same conditions the EDs0 of norepinephrine on the normal contraction was reduced. It is concluded that both reserpine pretreatment and hypothermia induce supersensitivity to the inotropic effects of norepinephrine by enhancing the cellular store of activator calcium while not affecting the ability of norepinephrine to release activator calcium. Hypothermia- and reserpine-induced supersenstitivity Potentiated contraction
Norepinephrine
1. I n t r o d u c t i o n
p e r f o r m e d . While t h e r e are several theories c o n c e r n i n g h y p o t h e r m i a - i n d u c e d supersensitivity in response t o s y m p a t h o m i m e t i c drugs, t h e p h e n o m e n o n p r o b a b l y occurs in t h e series o f events c o n s e q u e n t to t h e stimulation o f a d r e n o c e p t o r s b u t n o t in the r e c e p t o r binding site itself ( R e i n h a r d t et al., 1972; E n d o h et al., 1975). C o n s i s t e n t with this h y p o t h e s i s , Reiter and Stickel ( 1 9 7 0 ) have d e m o n s t r a t e d t h a t t h e d o s e - - r e s p o n s e curves for b o t h symp a t h o m i m e t i c agents and calcium are shifted to t h e right as t e m p e r a t u r e is raised. It is possible t h a t an altered utilization o f calcium b y m y o c a r d i a l cells m a y be the e v e n t responsible for t h e a p p a r e n t change in affinity o f s y m p a t h o m i m e t i c amines as t e m p e r a t u r e is lowered. Chronic p r e t r e a t m e n t with reserpine, an alkaloid k n o w n t o d e p l e t e c a t e c h o l a m i n e f r o m the heart, can i n d u c e supersensitivity t o t h e c h r o n o t r o p i c effects o f b o t h calcium and
Supersensitivity has b e e n d e f i n e d as t h e p h e n o m e n o n i n which t h e a m o u n t o f a substance r e q u i r e d t o p r o d u c e a given biological response is less t h a n n o r m a l ( F l e m i n g et al., 1 9 7 3 ) . This p h e n o m e n o n is n o r m a l l y i n d u c e d b y e i t h e r surgical or p h a r m a c o l o g i c a l denervat i o n o f the e f f e c t o r tissue and is t h o u g h t to reflect a c o m p e n s a t o r y change in the postj u n c t i o n a l e f f e c t o r cell in response to the subsequent, c h r o n i c loss o f n e u r o t r a n s m i t t e r (Fleming, 1 9 7 6 ) . R e c e n t l y , h o w e v e r , supersensitivity to the c h r o n o t r o p i c ( T r e n d e l e n b u r g , 1 9 6 8 ; Opperm a n n et al., 1 9 7 2 ; WSppel and T r e n d e l e n burg, 1 9 7 3 ) and i n o t r o p i c ( R e i n h a r d t et al., 1 9 7 2 ; B r o a d l e y , 1974) effects o f s y m p a t h o m i m e t i c agents has been achieved in m y o cardial p r e p a r a t i o n s b y m e r e l y lowering the t e m p e r a t u r e at which an e x p e r i m e n t is to be
Calcium
360
catecholamines (Fleming, 1976). In contrast, pretreatment with reserpine was thought not to induce supersensitivity to the inotropic effects of catecholamines and calcium (Taylor et al., 1974). Broadley and Lumley (1977) have reported, however, that reserpine pretreatment does induce a selective or specific supersensitivity to the inotropic effects of isoproterenol and salbutamol. As Broadley and Lumley were unable to demonstrate supersensitivity to the inotropic effects of either calcium or histamine, they concluded that this "selective" supersensitivity might be due to either an increased efficacy or proliferation of the fi-receptor. More recently, Duncan and Broadley (1977) have demonstrated that the selective supersensitivity induced by reserpine pretreatment to the inotropic effects of isoproterenol was masked if the temperature was lowered from 38 to 30 or 25°C. These authors speculated that both reserpine-induced and hypothermiainduced supersensitivity might result due either to an increase in the fi-receptor population or the relationship between receptor occupation and the response. However, Tenner and Carrier (1978) have reported that supersensitivity to the inotropic effects of calcium can be demonstrated in the electrically driven rabbit atria following reserpine pretreatment. This supersensitivity could be demonstrated at both 37 and 30°C without an apparent masking effect of the hypothermia-induced supersensitivity. As calcium may play a crucial role in both reserpine-induced and hypothermia-induced supersensitivity phenomena, it was decided that a study of these two forms of supersensitivity to the inotropic effects of norepinephrine and calcium might prove fruitful. One means of determining the utilization of calcium by cardiac tissue is by studying "potentiated" contractions on the first beat following a period of quiescence (Wood et al., 1969). Langer et al. (1975) noted that the potentiated contraction was a measure of the degree of activation of the myocardial cell and, therefore, a measure of the relative
T.E. TENNER, JR. ET AL.
amount of calcium released to the myofilaments. It was decided therefore to compare potentiated contractions obtained with left atria from untreated and reserpine-pretreated rabbits following various concentrations of norepinephrine and calcium at various temperatures in the hope of further illustrating the role of calcium in supersensitivity phenomena.
2. Materials and methods
2.1. Tissue preparation and procedures New Zealand white rabbits of either sex weighing between 1.0 and 1.5 kg were either chronically pretreated (7 days) with daily intramuscular injections of 0.1 mg/kg reserpine (Serpasil-CIBA) or were used as untreated controls. 24 h after the last pretreatment, the rabbits were stunned by a blow on the head and exsanguinated. After thoracot o m y , the hearts were excised, and immediately placed in oxygenated Chenoweth-Koelle solution (CKS) of the following composition (in mM): NaC1 120; KC1 5.63; CaC12 2.0; dextrose 9.7; MgC12 2.0; NaHCO3 25.0. Following separation from the ventricles, the left atrium was m o u n t e d vertically in a tissue chamber containing 5 0 m l of CKS which was continuously oxygenated with a gas mixture of 95% 02--5% CO2. The pH of the solution was maintained at 7.3 + 0.05 and the temperature was held at either 30 or 37°C depending upon the experiment to be performed. One portion of the atrium was secured to a Plexiglas electrode apparatus modified from that developed by Blinks (1966). The other portion was connected via a thin chain to a Grass FT-03 strain gauge transducer. The atrium was allowed to equilibrate for 45 min under a mechanically applied diastolic tension of 2 g. During equilibration, the bathing solution was completely changed every 15 min. The left atrial preparations were quiescent unless excited electrically b y the electrodes.
CALCIUM AND CARDIAC SUPERSENSITIVITY Two platinum wires were situated such that the Plexiglas clamp secured the lower portion of the atrial tissue against the heads of the electrodes. Preparations which beat spontaneously were discarded. Electrical stimulation was applied by a Grass SD-5 stimulator with a duration of 3 msec at a voltage approximately twice the threshold voltage (1--2 V). Preliminary studies using propranolol and atria from animals treated 24 h previously with reserpine indicated that this intensity did not result in significant release of endogenous neurotransmitter. The frequency of stimulation during the equilibration period and the subsequent experiments was 1.6 Hz. Isometric tension was recorded on a Grass Model 7 polygraph. For the purposes of this study, isometrically developed contractile force will be referred to as developed force. The developed force recorded at the end of the equilibration period will be referred to as the Basal Developed Force (BDF).
2.2. Experimental design Dose--response relationship for norepinephrine (L-arterenol, Sigma) were obtained using atria from reserpine-pretreated rabbits and untreated controls at 30 and 37°C. Following the addition of a given dose of norepinephfine, the atria were allowed to achieve a new steady state developed force. Upon achieving the plateau force, the stimulator was turned off and a 30 sec period of quiescence ensued. Upon resumption of stimulation, the first beat following the quiescent period, i.e. the potentiated contraction was recorded. A period of 30 sec was chosen as the amplitude of the potentiated contraction was found to be maximal following this quiescent period for atria tested at both 30 and 37°C. This observation is in agreement with that of Spikler and Cervoni (1969). Upon reattaining the steady state developed force achieved prior to the quiescent period, a higher dose of norepinephrine was added. This process continued until the maximal developed force (MDF) and maximal amplitude of the po-
361 tentiated contraction were achieved. Only one dose--response relationship was obtained for each atria. Potentiated contractions were also obtained for atria at three different calcium concentrations (0.6, 2.0 and 8.0 raM). After equilibration in normal CKS, the bathing medium was changed to a CKS containing the desired calcium concentration. The atria were allowed to achieve a new steady state developed force prior to obtaining potentiated contractions. These responses were obtained in a similar manner as those for norepinephrine.
2.3. Analysis and statistics The presence of supersensitivity was determined by comparison of geometric mean EDs0 values (Fleming et al., 1973; Fleming et al., 1972). The EDs0 values for norepinephrine were obtained graphically from the individual dose--response curves following conversion of the absolute response at each concentration to percentages of maximum response. The EDs0 values were then compiled to yield the geometric mean -+ 95% confidence interval. In addition, estimates of the change in sensitivity to norepinephrine are described as the ratio of the geometric mean EDs0 values. All other data are absolute values presented as arithmetic means-+ S.E.M. Statistical significance was determined by use of the Student's t-test. Differences were considered to be significant if P < 0.05.
3. Results Fig. 1 illustrates the influence of hypothermia (decreasing the temperature of the bathing medium from 37 to 30°C) on norepinephrine-induced increases in developed force in left atria from untreated rabbits. At subthreshold doses of norepinephrine (10 -9 M), a hypothermia-induced increase in basal developed force (BDF) was apparent. In contrast, the maximum developed force (MDF) ohtained to high doses of norepinephrine was
IEDs° i
362 6.0 __
5.0
v
4.0
30.0
T.E. TENNER, JR. ET AL. 7.0 60 50
~°°I
O
30
22.0
40
14.0
30
ED50
o >
X
20
,
C~
~
1
I 9
i 8
I 7
i 6
z 5
i 4
30oC
37oc
9
Fig. 1. The influence of temperature on the dose-response relationship and Geometric Mean EDs0 value for norepinephrine. Open circles represent responses obtained at 30°C while the open triangles represent responses obtained at 37°C. Each point represents the mean ± S.E.M. of 6--8 experiments. Vertical bars on the EDs0 values represent the 95% confidence interval for the Geometric Means.
not significantly altered by the temperature at which the experiment was performed. When the EDs0 values for norepinephrine were calculated for the two temperatures, bY" pothermia-induced supersensitivity was apparent. The EDs0 value for norepinephrine at 30°C was approximately one third of that at 37°C indicating a threefold increase in sen-
5.0
±
.oofi ED50
4.0 ,2°
~,
3.0 -$8
x
20
22.0 14.0
Z 1.0
6.0 I
I
I
9
8
7
-Log ENE]
10 J
-Log [NE] (M}
>
×
2.0
6.0
(~1 I
I
I
6
5
4
r~ 37%
37~C +RES.
Fig. 2. The influence of reserpine pretreatment on the dose-response relationship and Geometric Mean EDs0 value for norepinephrine when tested at 37°C. Open triangles represent untreated controls while closed triangles represent atria from reserpine-pretreated rabbits. Each point represents the mean ± S.E.M. of 6--8 experiments. Vertical bars on the EDs0 values represent the 95% confidence interval for the Geometric Means.
22.0
o
I 8
I 7
I 6
I 5
I 4
°°nh 14.0
30oC
30cC +RES.
- Lo9 [NE] (MI
Fig. 3. The influence of reserpine pretreatment on the dose-response relationship and Geometric Mean EDso value for norepinephrine when tested at 30°C. Open circles represent untreated controls while closed circles represent atria from reserpine-pretreated rabbits. Each point represents the mean ± S.E.M. o f 8-10 experiments. Vertical bars on the EDs0 values represent the 95% confidence interval for the Geometric Means.
sitivity at the lower temperature. Fig. 2 illustrates the influence of reserpine pretreatment upon norepinephrine-induced increases in developed force when the experiments were performed at 37°C. In this group of experiments, neither the BDF or the MDF of atria from reserpine-pretreated rabbits were significantly greater than those from untreated animals. While the BDF was increased, significance was not obtained in contrast to a previous report by Tenner and Carrier (1978). When the EDs0 values obtained at 37°C were compared, the value for atria from reserpine-pretreated animals was approximately one third of that from untreated controls. This would indicate a reserpine-induced supersensitivity as the sensitivity of atria from pretreated animals was threefold greater than untreated controls. It is interesting to note that the EDs0 values obtained for atria from untreated rabbits at 30°C and those for atria from reserpine-pretreated rabbits at 37°C were not significantly different from each other (figs. 1 and 2). Fig. 3 illustrates the effects of reserpine pretreatment upon norepinephrine-induced
CALCIUM AND CARDIAC SUPERSENSITIVITY increases in developed force when the experiments were performed at 30°C. In this group of experiments, both the BDF and MDF were greater in atria from reserpine-pretreated rabbits than from untreated controls. In contrast, however, the EDs0 values for atria of untreated and reserpine-pretreated animals obtained at 30°C were not significantly different. It is apparent that hypothermiainduced supersensitivity and reserpine-induced supersensitivity were not additive, i.e. the EDs0 values for atria from reserpine-pretreated rabbits were not significantly different when obtained at 37 and 30°C (figs. 2 and 3). As stated earlier, Tenner and Carrier (1978) reported that reserpine-induced supersensitivity to calcium was not masked by hypothermia-induced supersensitivity. It was therefore concluded that the norepinephrine-induced increases in developed force might be dependent upon the utilization of calcium as influenced by either hypothermia or reserpine pretreatment. It was of interest then to examine calcium utilization by the atrial preparations under the various experimental conditions. The potentiated contraction (the first beat obtained following a thirty second period of quiescence) was chosen as a measure of the relative a m o u n t of calcium released to the myofilaments (Langer et al., 1975). It was first necessary to demonstrate the dependence o f the potentiated contraction amplitude on the extracellular calcium concentration. Figure 4 illustrates this relationship. The amplitude of the potentiated contraction also depends upon the temperature and whether or not the atria used are from reserpine-pretreated or untreated rabbits. As is obvious from fig. 4, the potentiated contraction amplitude obtained with 2.0 mM calcium at 37°C < 37°C + reserpine pretreatment < 30°C < 30°C + reserpine pretreatment. In other words, atria from reserpine-pretreated animals produced amplitudes greater than untreated controls at each of the temperatures studied. Fig. 5 illustrates the influence of temperature on the amplitude of the potentiated contraction obtained using various doses of
363
8.0
7,0 -~
i
6.0
tJ
4.0
"5
3.0
~
2.0 1.0
30°C +RES,
30oC
37°C ÷RES
37oC
Fig. 4. The influence of calcium on the amplitude of the potentiated contraction obtained with atria from either reserpine-pretreated or untreated rabbits at both 30 and 37°C. (~) 0.2 mM, (..) 2.0 mM, (~) 8.0 mM CaC12. Vertical bars represent the S.E.M. of 11-14 experiments at each calcium concentration.
norepinephrine. Note that at subthreshold doses of norepinephrine, the hypothermiainduced increase in the potentiated contraction amplitude, as evidenced, in fig. 4, was
8.0 7.0 6.0
I '5
5.o
i ~
4.0
~
30
6
20 1.0
9
8
7
6
5
4
-Log [NE] (M)
Fig. 5. The influence of temperature on the amplitude of the potentiated contraction obtained for various doses of norepinephrine using atria from untreated rabbits. (~) represents atria tested at 30°C while (•) represents atria tested at 37°C. Vertical bars represent the S.E.M. of 6--8 experiments.
364
T.E. TENNER, JR. ET AL. 6.0
8.0
50
70
i iili
4o U~
3.0
5
20
g ~_
1.0
9
8
7
6
5
6.0
5.0 4.0 30
4
- L o g [NE] {M)
Fig. 6. The influence of reserpine on the amplitude of the potentiated contraction obtained for various doses of norepinephrine at 37°C. (~) represents atria from reserpine-pretreated rabbits while (a) represents atria from untreated rabbits. Vertical bars represent the S.E.M. of 6--8 experiments.
apparent. While norepinephrine increased the amplitude of the potentiated contraction when studied at 37°C, it had no significant effect on the amplitude of potentiated contractions obtained at 30°C. It is important to remember that norepinephrine increased developed force at both temperatures studied (fig. 1). When studied at 37 ° C, atria from reserpinepretreated rabbits produced potentiated contractions of significantly greater amplitude than controls (fig. 6). In a similar fashion as observed for atria studied at 30°C, norepinephrine did not further increase the amplitude of the potentiated contractions of atria from reserpine-pretreated animals, while increasing that for atria from control animals. At a temperature of 30°C, atria from reserpine-pretreated rabbits developed potentiated contractions of significantly greater amplitude than controls (fig. 7). Norepinephrine did not further increase the amplitude of the potentiated contractions of atria from either control or reserpine-pretreated rabbits under the hypothermic condition. There would appear to be a correlation between the EDs0 values reported in figs. 1, 2, and 3 and the ability of norepinephrine to alter the amplitude of the potentiated con-
o-
2.0
H
1.0
i 9
V//A I 8
7
6
5
-Log ~NE] ',M)
Fig. 7. The influence of reserpine on the amplitude of the potentiated contraction obtained for various doses of norepinephrine at 30°C. ([~) represents atria from reserpine-pretreated rabbits while ([]) represents atria from untreated rabbits. Vertical bars represent the S.E.M. of 8--10 experiments.
traction. Reserpine pretreatment and hypothermia resulted in a decrease in the ED~0 values obtained for norepinephrine thus indicating supersensitivity. These two experimental conditions also produced an increase in the potentiated contraction such that no further increase was produced by norepinephrine.
4. Discussion The present study has demonstrated that both chronic reserpine pretreatment and hypothermia will induce supersensitivity, as determined by geometric mean EDs0 values, to the inotropic effects of norepinephrine. The supersensitivity was associated with those experimental conditions where the amplitude of the potentiated contraction could not be further enhanced by norepinephrine. Recently, Duncan and Broadley (1977) published results similar to those described in the present paper. These authors reported that hypothermia could mask the supersensitivity induced by reserpine pretreatment to the inotropic effects of isoproterenol and sal-
CALCIUM AND CARDIAC SUPERSENSITIVITY butamol. In an earlier study, Broadley and Lumley {1977) proposed that the supersensitivity induced by chronic reserpine pretreatment was selective for fi-agonists as they could not demonstrate supersensitivity to either calcium or histamine. It was suggested that the mechanism of action for this selective supersensitivity may result from either an increase in the fi-receptor population or a change in the relationship between receptor occupation and response. Duncan and Broadley (1977) suggested that these two explanations might also apply to the mechanism of hypothermiainduced supersensitivity as it had the ability to mask the supersensitivity induced by reserpine pretreatment. In contrast, Tenner and Carrier (1978) reported that supersensitivity to the inotropic effects of calcium could be induced by chronic reserpine pretreatment and by hypothermia. In addition, reserpine-induced supersensitivity to the inotropic effect of calcium was not masked by that induced by hypothermia. It would appear, therefore, that these two experimental conditions (reserpine pretreatment and hypothermia) induce supersensitivity to the inotropic effects of calcium through different mechanisms as their effects on sensitivity are aditive i.e. neither masks the effects of the other (Tenner and Carrier, 1978). On the other hand, based on the data of the present study as well as that of Duncan and Broadley (1977), reserpine pretreatment and hypothermia appear to induce supersensitivity to the inotropic effects of norepinephrine via the same mechanism i.e. hypothermia masks the effects of reserpine pretreatment and vice versa. One explanation for these results could be that reserpine pretreatment and hypothermia induce supersensitivity to the inotropic effects of calcium via different alterations of calcium utilization (Tenner and Carrier, 1978) while the supersensitivity to norepinephrine results from some common change produced in the fl-receptor (Broadley and Lumley, 1977; Duncan and Broadley, 1977). Another possibility is that reserpine
365 pretreatment and hypothermia influence different steps in the sequence of events resulting in excitation--contraction coupling, and that the inotropic effect of norepinephrine is enhanced in the same manner by both influences. In order to test the latter hypothesis an index of calcium utilization of cardiac muscle was required. Langer et al. (1975)stated that the magnitude of the first beat following a period of quiescence was a measure of the degree of activation of the muscle and therefore of the relative amount of calcium released to the myofilaments. In the present study the amplitude of the potentiated contraction was quantitated and used as an index of calcium utilization. Wood et al. (1969) concluded that the amplitude of the potentiated contraction dependend upon the presystolic level of intracellular calcium bound to specific (rapid release) sites and that this amount of activator calcium was predetermined by the frequency of stimulation, the temperature, the extracellular calcium concentration, and the elapsed time since termination of the last absolute refractory period (i.e. the period of quiescence). Wood et al. postulated that with each depolarization, "the amount of calcium ions dissociated from rapid release sites in the sarcoplasmic reticulum and other intracellular membrane surfaces.., is a relatively constant fraction of the total quantity of calcium stored at these release sites. The greater the amount of calcium present in these structures, the greater the amount dissociated to intracellular free calcium ions by a given action potential". As indicated by increases in the amplitude of potentiated contractions obtained in the present study, reserpine pretreatment and hypothermia both appear to increase the functional amount of activator calcium stored for future release to the myofilaments. Reserpine pretreatment probably increases this store through a different mechanism than hypothermia as an "additive" effect upon the amplitude of the potentiated contraction was observed at each temperature studied. This
366
hypothesis is consistent with the data of Tenner and Carrier (1978) in that reserpineinduced supersensitivity to calcium could be demonstrated under hypothermic conditions. According to Wood et al. (1969), a certain fixed fraction of this store of activator calcium is released for the development of force with each depolarization. As this store is increased b y either reserpine pretreatment, hypothermia, or both, the amount of activator calcium released with each depolarization is increased even though the fraction released remains fixed. This could account for the increases in BDF achieved with hypothermic conditions and following chronic reserpine pretreatment. It should be noted that while atria from reserpine pretreated animals tested at 37°C displayed a greater BDF than untreated controls, the difference was not significant. This finding is interpreted as an artifact of the given experiments as a significant increase in BDF was observed for reserpine pretreated atria at 37°C in other experiments testing calcium (Tenner and Carrier, 1978). The ability of norepinephrine to produce a positivie inotropic effect lies in several postulated mechanisms. Norepinephrine is thought to increase transmembrane calcium influx (Rasmussen et al., 1972; Meinertz et al., 1973), increase sequestration of calcium (Katz and Repke, 1973) and increase the release of stored activator calcium. (Sinebourne and White, 1970; Fabiato and Fabiato, 1975). Increasing calcium influx and sequestration would result in increasing the size of the stored calcium pool. If reserpine pretreatmerit or hypothermia increased the amount of activator calcium available for release, the importance of these two latter mechanisms would be diminished. On the other hand, each dose of norepinephrine would increase the a m o u n t of stored calcium released to a greater extent than in untreated atria at 37°C. At 37°C, each dose of norepinephrine would have to increase the size of the activator calcium pool by increasing calcium influx and sequestration as well as increase the release of this growing calcium pool. This hypothesis
T.E. TENNER, JR. ET AL.
is consistant with the results of the present study. Norepinphrine was able to increase the amplitude of the potentiated contraction only in untreated atria at 37 ° C. On the other hand, no significant increase in the amplitude of the potentiated contraction was observed with norepinephrine in atria from either reserpine pretreated animals or in untreated atria studied at 30°C. Because reserpine pretreatment and hypothermia increased the amount of calcium available for release, each dose of norepinephrine could release more calcium than was possible for the same dose in untreated atria at 37°C. This would explain why the EDs0 values for reserpine pretreated atria at 37 and 30°C were the same as that for untreated atria at 30°C as well as w h y these EDs0 values were significantly less than that for untreated atria at 37 ° C. It would appear therefore that the mechanism by which reserpine pretreatment and hypothermia induce supersensitivity could be by increasing the functional amount of activator calcium which can be released to the myofilaments and not by altering the ability of norepinephrine to release that calcium. This conclusion is consistant with the observation that supersensitivity to the inotropic effect of calcium is induced by reserpine pretreatment and hypothermia in an additive fashion (Tenner and Carrier, 1978).
Acknowledgements This work was supported in part by the National Institutes of Health (NIH) Grant No. HL17899, AFOSR Grant No. 76-3029, and the British Columbia Heart Foundation.
References Blinks, J.R., 1966, Field stimulation as a means of effecting the graded release of autonomic transmitter in isolated heart muscle, J. Pharmacol. Exptl. Therap. 151,221. Broadley, K.J., 1974, The interpretation of doseresponse curves expressed by various graphical means to determine the effects of temperature
CALCIUM AND CARDIAC SUPERSENSITIVITY on the resonses of guinea-pig isolated atria to isoprenaline, Arch. Intern. Pharmacodyn. 210,147. Broadley, K.J. and P. Lumley, 1977, Selective reserpine-induced supersensitivity of the positive inotropic and chronotrpic responses to isoprenaline and slabutam01 in guinea-pig isolated atria, Brit. J. Pharmacol. 59, 51. Duncan, C. and K.J. Broadley, 1977, The influence of temperature upon reserpine-induced supersensitivity of guinea-pig isolated atria to isoprenaline and salbutamol, Naunyn-Schmiedeb. Arch. Pharmacol. 297,163. Endoh, M., J. Wagner and H.J. Schiimann, 1975, Influence of temperature on the positive inotropic effects mediated by ~-,/3-adrenoceptors in the isolated rabbit papillary muscle, Naunyn-Schmiedeb. Arch. Pharmacol. 287, 61. Fabiato, A. and F. Fabiato, 1975, Relaxing and inotropic effects of cyclic AMP on skinned cardiac cells, Nature 253,556. Fleming, W.W., 1976, Variable sensitivity of excitable cells: Possible mechanisms and biological significance, Rev. Neurosci. 2, 43. Fleming, W.W., J.J. McPhillips and D.P. Westfall, 1973, Postjunctional supersensitivity and subsensitivity of excitable tissues to drugs, Ergeb. Physiol. 68, 55. Fleming, W.W., D.P. Westfall, I.S. De La Lande and L.B. Jehlett, 1972, Log-normal distribution of equieffective doses of norepinephrine and acetylcholine in several tissues, J. Pharmacol. Exptl. Therap. 1 8 1 , 3 3 9 . Katz, A.M. and K.I. Repke, 1973, Calcium membrane interactions in the myocardium: Effects of ouabain, epinephrine, and 3',5'-cyclic adenosine monophosphate, Amer. J. Cardiol. 31,193. Langer, G.A., S.D. Serena and L.M. Nudd, 1975, Localization of contractile dependent calcium: comparison of manganese and verapamil in cardiac and skeletal muscle, Amer. J. Physiol. 229, 1003. Meinertz, T., H. Naurath and H. Scholz, 1973, Stimulatory effects of DB-c-AMP and adrenaline on myocardial contraction and ~SCa exchange. Experiments at reduced concentration and low frequencies of stimulation, Arch. Pharmacol. 279, 327. Oppermann, J.A., C.F. Ryan and C.O. Haavik, 1972,
367 The role of metabolism in temperature-dependent supersensitivity of guinea-pig atria to sympathomimetic amines, European J. Pharmacol. 18,266. Rasmussen, H., D.B.P. Goodman and A. Tenenhouse, 1972, The role of cyclic AMP and calcium in cell activation, Crit. Rev. Biochem. 1, 95. Reinhardt, D., J. Wagner and H.J. Schiimann, 1972, Influence of temperature on the sensitivity of the /3-receptors and the contractility of guinea-pig atrium, Naunyn Schmiedeb. Arch. Pharmacol. 275, 95. Reiter, M. and F.J. Stickel, 1970, Zur Frage einer therapeutischen Steigerung der Kontraktilit/it bei Abkfihlung des Herzens, Klin. Wschr. 4 8 , 9 3 5 . Shinebourne, E. and R. White, 1970, Cyclic AMP and calcium uptake of the sarcoplasmic reticulum in relation to increased rate of relaxation under the influence of catecholamines, Cardiovasc. Res. 4, 194. Spikler, B. and P. Cervoni, 1969, Electrical membrane activity and frequency-force relations in normal, denervated and reserpine pretreated cat heart muscle, J. Pharmacol. Exptl. Therap. 168, 60. Taylor, D.A., D.P. Westfall and W.W. Fleming, 1974, The effect of reserpine treatment on the chronotropic and inotropic sensitivities of the perfused guinea-pig heart to norepinephrine and calcium, European J. Pharmacol. 29, 102. Tenner, T.E. and O. Carrier, 1978, Reserpine-induced supersensitivity to the chronotropic and iontropic effects of calcium in rabbit atria, J. Pharmacol. Exptl. Therap. 205,183. Trendelenburg, U., 1968, The effect of cocaine on the pacemaker of isolated guinea-pig atria, J. Pharmacol. Exptl. Therap. 161,222. Wood, E.H., R.L. Heppner and S. Weidmann, 1969, Inotropic effects of electric currents. I. Positive and negative effects of constant electric currents or current pulses applied during cardiac action potentials. II. Hypothesis: calcium movements, excitation-contraction coupling and inotropic effects, Circulation Res. 24,409. WSppel, W. and U. Trendelenburg, 1973, Temperature-dependent supersensitivity of isolated atria to catecholamines, European J. Pharmacol. 23,302.