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INHALATION ANAESTHETICS DECREASE CALCIUM CONTENT OF CARDIAC SARCOPLASMIC RETICULUM†
Br. J. Anaesth. (1989), 62, 669-673
INHALATION ANAESTHETICS DECREASE CALCIUM CONTENT OF CARDIAC SARCOPLASMIC RETICULUMf M. KATSUOKA AND S. T. OHNISHI
MATERIALS AND METHODS
Langendorff perfusion model MASAVUKI KATSUOKA, M.S.; S. TSUYOSHI OHNISHI, PH.D.;
Membrane Research Institute, University City Science Center, 3401 Market Street, Philadelphia, Pa 19104 U.S.A. Accepted for Publication: December 6, 1988. fPreliminary results were presented at the Annual Meeting of the Biophysical Society at Phoenix, Arizona (March, 1988).
Male Sprague-Dawley rats (weight 300-400 g) were anaesthetized by i.p. injection of pentobarbitone 50 mg kg"1. The heart was removed quickly and immersed in ice cold Krebs-Henseleit bicarbonate solution (KHB solution; content
Downloaded from http://bja.oxfordjournals.org/ at University of Bath Library & Learning Centre on June 22, 2015
It is known that general anaesthetics markedly reduce myocardial contractility [1-4]. In in vitro SUMMARY experiments, Price [5,6] found that anaesthetic- In a Langendorff perfused rat heart model, induced reduction in contractile force of papillary caffeine was shown to have a negative inotropic muscle was antagonized by increasing calcium effect similar to that of inhalation anaesthetics. concentration in the bathing medium. One of the Both inhalation anaesthetics and caffeine desites of action suggested by several investigators creased calcium content of left ventricular muscle was the plasma membrane. Langer, Serena and as measured by atomic absorption spectroscope. Nudd [7] suggested that membrane surface- Halothane 2.8% decreased left ventricular pressbound calcium plays a role in the contraction of ure by 75.4 (SEM 4.4)% and decreased the cardiac muscle. Using a La3+ exchange technique, calcium content by 30%. Enflurane 5.1% deOhnishi and colleagues [8] have shown that creased the pressure by 72.4 (6.0) % and dehalothane reduces the amount of La3+-exchange- creased calcium content by 31%. Both halothane able calcium in trabecular muscle in proportion to and enflurane decreased the calcium content of the decrease in contractility. They showed that sarcoplasmic reticulum (SR) as measured by the calcium binding of the preparation from caffeine-induced calcium release. There was a cardiac plasma membrane lipoprotein was in- high degree of correlation between the negative creased in the presence of general anaesthetics and inotropic effect of inhalation anaesthetics and ethanol [9]. the decrease in calcium content of the SR While calcium bound to the plasma membrane (x = 0.95 for halothane, r = 0.91 for enflurane). may play a role in cardiac muscle contraction, the These data suggest that inhalation anaesthetics major site of interest is the sarcoplasmic reticulum increase the calcium permeability of cardiac SR, (SR). Su and Kerrick [10] showed that halothane thereby decreasing the calcium content of SR. caused myocardial depression by inhibiting cal- resulting in the negative inotropic effect. cium uptake of SR in skinned fibres. Ohnishi [11,12] found that both halothane and caffeine increased the calcium-induced calcium release from the SR. Using SR prepared from malignant using a Langendorff perfused rat heart model. hyperthermia-susceptible skeletal muscle, he We measured both the total calcium content of the found that both caffeine and halothane increased left ventricular muscle with atomic absorption the calcium permeability of the SR [13]. We spectroscopy and the amount of calcium ion postulated that halothane may also increase released from the SR using a caffeine-induced calcium permeability of the cardiac SR, thereby calcium release method [14, 15]. decreasing the calcium content of the SR. In the present study, we have tested this hypothesis
BRITISH JOURNAL OF ANAESTHESIA
670 Perfusate •
Anaesth. gas
Ca2*-free' buffer
Ca2+-free buffer + caffeine
Control
o o Enflurane
1min
1s
FIG. 1. Caffeine-induced contraction: Control (no addition); effect of 2.8% halothane and effect of 5.1 % enflurane. The heart was perfused with inhalation anaesthetic (Anaesth.) for a few minutes and with Ca2+-free buffer for 30 s, then with Ca2+-free buffer containing caffeine 40 mmol litre"1.
(mmol litre"1): NaCl 118, KC1 4.7, CaCl2 1.25, MgSO4 1.25, KH2PO4 1.25, NaHCO3 24, glucose 15; pH adjusted to 7.4 when equilibrated with 5 % carbon dioxide in oxygen at 37 °C). Extraneous tissue was removed and the aorta was cannulated on the perfusion apparatus. The left ventricular pressure was measured with a latex balloon connected to a Statham PB 23 transducer via a thin catheter. The balloon was inserted through the mitral valve and filled with water (volume 0.1-0.2 ml).
at 2000 rev min"1 for 10 min and the calcium concentration of the supernatant measured using a polarized Zeeman effect atomic absorption spectrophotometer (Hitachi Z-8000). The pellet was dissolved in 30% potassium hydroxide 10 ml and the protein concentration determined by a biuret method. Measurements were made in triplicates. Estimation of calcium content of sarcoplasmic reticulum using caffeine
Calcium content of SR was measured by the procedures reported by Endo [14,15] and Heide and colleagues [16], with a small modification. The heart was stimulated continuously (5 Hz). The perfused heart was stimulated by electric After equilibration with KHB solution for pulses (10 V, 4-5 Hz, 10 ms duration). The KHB 15 2+min, the perfusing solution was switched to a solution was changed to a KHB solution con- Ca -free KHB2+ solution for 30 s then changed solution containing taining either caffeine or anaesthetic agents (the again, to a Ca -free KHB 1 latter vaporized from a Drager vaporizer and caffeine 40 mmol litre" . Endo found that the bubbled into the solution) and perfused for calcium release at this high concentration of lOmin. The pressure decreased and reached a caffeine represented the amount of calcium left in new plateau value within the first 5 min. The per- the SR [14,15]. Several contractions of the heart fusing solution was changed to a Ca2+-free KHB were observed (fig. 1) and the height of each solution, and perfused for 1 min. The left ven- contraction measured. It was assumed that the tricular muscle was cut into small pieces with height of each contraction was related to calcium scissors and homogenized with a polytron hom- release from the SR and thus the summation of ogenizer (volume 10 ml). The homogenate was each contraction was regarded as the total calcium SR in the presence of caffeine extracted with HC1 2 mol litre"1 for 24 h with release from the 1 constant shaking. The suspension was centrifuged 40 mmol litre" .
Relationship between left ventricular developed pressure (LVDP) and calcium content of the left ventricular muscle
Downloaded from http://bja.oxfordjournals.org/ at University of Bath Library & Learning Centre on June 22, 2015
Halothane
INHALATION ANAESTHETICS AND THE HEART Determination of anaesthetic concentration
671
100-1
Concentrations of halothane and enflurane from the respective vaporizers were determined by gas chromatography. a 504
Data analysis
Each datum point was obtained from six separate experiments. Statistical analysis was performed using Student's t test. RESULTS
2
Negative inotropic effects of caffeine, halothane and enflurane
When the heart was perfused with caffeine, diastolic pressure increased. Systolic pressure increased immediately and transiently, then decreased to a new plateau value which was smaller than the original one (negative inotropic effect). Similar changes were observed with both halothane and enflurane (fig. 2). LVDP was depressed by these agents in a dose-related manner (fig. 3). A reduction of 50% was produced by 1.8% halothane and 4.0 % enflurane.
H
H
Caffeine (mmol litre"1) 4 8
ra
10 Anaesthetic gas (7.) FIG. 4. Effects of caffeine ( # ) , halothane (O) and enflurane (A) on calcium content of left ventricular muscle (mean and SEM) (n = 6). Calcium content was measured by atomic absorption spectrophotometry. Significant difference from normal perfusate: * P < 0.05; ** P < 0.01. Halothane
Caffeine
1
6 10 Anaesthetic gas (%) FIG. 3. Effects of caffeine ( # ) , halothane (O) and enflurane (A) on the developed pressure (LVDP) expressed as a percentage of contraction perfused with buffer solution without agents (mean and SEM) (n = 6).
Effects of caffeine, halothane and enflurane on calcium content of left ventricular muscle
All these agents decreased the calcium content of left ventricular muscle as measured by atomic absorption spectrophotometry (fig. 4). When the heart was perfused with 2.8% halothane, 5.1% enflurane or caffeine 2 mmol litre"1, the calcium content was decreased to 70.3 (9.5) % (P < 0.05), 69.4 (6.9)% (P<0.05) and 56.1 (6.9)%
—I
Enflurane
b>4o
e CD
1% Halothane
u | '_o 03
O
21 Enflurane
1s
1min
FIG. 2. Effects of caffeine 4 mmol litre" 1 , 1 % halothane and 2 % enflurane on the left ventricular developed pressure (LVDP) of the isolated heart. Arrows indicate change in perfusate.
50 LVDPI/J)
100
50 LVDP (7.)
100
FIG. 5. Relationship between left ventricular developed pressure (LVDP) and calcium content in left ventricular muscle: effects of halothane (r = 0.65, P < 0.05) and enflurane (r = 0.60, P < 0.05).
Downloaded from http://bja.oxfordjournals.org/ at University of Bath Library & Learning Centre on June 22, 2015
Caffeine (mmol litre'1) 4 8
0J 0
BRITISH JOURNAL OF ANAESTHESIA
672 100n
Halothane
Enflurane
503 T3
c
•i
0 50
100
50
100
LVDP (7.) LVDPU) a FIG. 6. Effect of halothane (r = 0.95, P< 0.001) and enflurane (r = 0.91, P < 0.001) on the relationship between left ventricular developed pressure (LVDP: percentage of control) and calcium content in the SR. Calcium content in the SR was measured as a caffeine-induced contraction and expressed as a percentage of control.
(P < 0.01) of control, respectively. The relationship between LVDP and the calcium content of the left ventricular muscle was similar with halothane and enflurane (r = 0.65, P < 0.05 and r = 0.60, P < 0.05, respectively) (fig. 5). Relationship between LVDP and calcium content in the SR In this experiment, calcium content was estimated by caffeine-induced calcium release from the SR (fig. 1). There was a correlation between LVDP and calcium content of the SR (r = 0.95, P < 0.001 and r = 0.91, P < 0.001 for halothane and enflurane, respectively) (fig. 6). DISCUSSION
Although inhalation anaesthetics are well known to reduce contractile force of the heart, the exact mechanism of action is not well understood. Using isolated skeletal muscle SR, it has been shown that inhalation anaesthetics, such as halothane and enflurane, stimulate calcium release from the SR [17]. It has also been demonstrated that both caffeine and inhalation anaesthetics increase the calcium permeability of SR membranes [11,12] and it is well known that caffeine causes calcium release from the SR [14-17]. These findings suggest that there are close similarities between the mechanism of action of caffeine and that of inhalation anaesthetics on the SR, and that the mechanism of negative inotropism may be related to the stimulated calcium release from the SR. Using a Langendorff perfused heart model, we attempted to test this hypothesis.
ACKNOWLEDGEMENTS This work was supported by NIH grants GM 33025 and GM 35681. We thank Dr Bryan Marshall, Department of Anesthesiology, University of Pennsylvania, for determining the halothane and enflurane concentrations.
REFERENCES 1. Brewster WR, Isaacs JP, Waing-Anderson T. Depressant effect of ether on myocardium of the dog and its modification by reflex release of epinephrine and norepinephrine. Journal of Pharmacology and Experimental Therapeutics 1953; 175: 399-414.
Downloaded from http://bja.oxfordjournals.org/ at University of Bath Library & Learning Centre on June 22, 2015
CD
First, we demonstrated that caffeine, a well known SR calcium releasing agent [14-17], has a negative inotropic effect on cardiac muscle (figs 2, 3). The action of caffeine and inhalation anaesthetics on the SR seemed to be similar: they caused an immediate increase in systolic pressure followed by a decrease, and they caused an increase in diastolic pressure (fig. 2). Second, we measured the calcium content of the left ventricular muscle using atomic absorption spectroscopy. Both inhalation anaesthetics and caffeine decreased the calcium content in a doserelated manner (fig. 4). Third, we used a method of estimating the calcium content of SR [14-16] and demonstrated that the tension developed in a muscle fibre in the presence of high concentrations of caffeine was related to the amount of calcium released from the SR. We applied the method to the heart by assuming that each contraction represented die amount of calcium released from cardiac SR. Caffeine-induced contraction of the heart was decreased with an increase in concentration of inhalation anaesthetic (fig. 1), suggesting, in agreement with results obtained by others [18,19], that inhalation anaesthetics decrease the calcium content of the SR. Several mechanisms may be involved in the negative inotropic effect of inhalation anaesthetics. They affect calcium binding of the plasma membrane [8,9,20] and the SR [10,18,19,2124], and the action potential [25,26]. Our data suggest the possibility of a new mechanism involving the myocardial SR. As postulated previously [27], inhalation anaesthetics increase the calcium permeability of the cardiac SR (and of skeletal SR). This may cause a decrease in calcium content of the SR resulting in a decrease in the maximum developed tension of heart muscle,— that is, negative inotropism.
INHALATION ANAESTHETICS AND THE HEART
673
Downloaded from http://bja.oxfordjournals.org/ at University of Bath Library & Learning Centre on June 22, 2015
2. Price HL, Helrich M. The effect of cyclopropane, diethyl 14. Endo M. Mechanism of action of caffeine on the ether, nitrous oxide, thiopental and hydrogen ion consarcoplasmic reticulum of skeletal muscle. Proceedings of centration on the myocardial function of the dog heartthe Japan Academy 1975; 51: 479-484. lung preparation. Journal of Pharmacology and Exper- 15. Endo M. Calcium release from the sarcoplasmic reticuimental Therapeutics 1955; 115: 206-216. lum. Physiological Reviews 1977; 57: 71-108. 3. Rusy BF, Komai H. Anesthetic depression of myocardial 16. Heide RSV, Altschuld RA, Lamka KG, Ganote CE. Modification of caffeine-induced injury in Caz+-free contractility: A review of possible mechanisms. Anesperfused rat hearts. American Journal of Pathology 1986; thesiology 1987; 67: 745-766. 123: 351-364. 4. Brown BR, Crout JR. A comparative study of the effect of five general anesthetics on myocardial contractility. Anes- 17. Ohnishi ST. Effects of halothane, caffeine, dantrolene and thesiology 1971; 34: 236-245. tetracaine on the calcium permeability of skeletal sarcoplasmic reticulum of malignant hyperthermic pigs. Bio5. Price HL. Calcium reverses myocardial depression caused chimica et Biophysica Ada 1987; 897: 261-268. by halothane: Site of action. Anesthesiology 1974; 218: 576-579. 18. Wheeler DM, Rice RT, Hansford RC, Lakatta EG. Halothane transiently increases free intracellular calcium 6. Price HL. Myocardial depression by nitrous oxide and its concentration in isolated adult rat heart cells. Anesreversal by Ca++. Anesthesiology 1976; 44: 211-215. thesiology 1987; 67: A94. 7. Langer GA, Serena SD, Nudd LM. Cation exchange in heart cell culture: Correlation with effects on contractile 19. Herland JS, Stephenson DG, Julian FJ. Halothane affects force. Journal of Molecular and Cellular Cardiology 1974; the contractile apparatus and sarcoplasmic reticulum of 6: 149-161. mechanically skinned rat ventricular fibers. Biophysical Journal 1988; 53: 335a. 8. Ohnishi ST, DiCamillo CA, Singer M, Price HL. Correlation between halothane-induced myocardial de- 20. Porsius AJ, van Zwieten PA. Influence of halothane on 3+ 2+ calcium movements in isolated heart muscle and in pression and decreases in La -displaceable Ca in isolated plasma membranes. Archives Internationales de cardiac muscle cells. Journal of Cardiovascular PharPharmacodynamie el de The'rapie 1975; 218: 29-39. macology 1980;2: 67-75. 9. Ohnishi ST, Obzansky DM, Price HL. The increase in 21. Bosnjak ZJ, Kampine JP. Effects of halothane on transmembrane potentials, Ca2+ transients, and papillary calcium binding of cardiac plasma membrane lipoprotein muscle tension in the cat. American Journal of Physiology caused by general anesthetics and alcohol. Canadian 1986; 251: H374-381. Journal of Physiology and Pharmacology 1980; 58: 525530. 22. Blanck TJJ, Thompson M. Enflurane and isoflurane stimulate calcium transport by cardiac sarcoplasmic 10. Su JY, Kerrick WGL. Effects of halothane on caffeinereticulum. Anesthesia and Analgesia 1982; 61: 142-145. induced tension transients in functionally skinned myocardial fibers. Pflugers Archiv. European Journal of 23. Casella ES, Suite NDA, Fisher YI, Blank TJJ. The effect Physiology 1979; 380: 29-34. of volatile anesthetics on the pH dependence of calcium uptake by cardiac sarcoplasmic reticulum. Anesthesiology 11. Ohnishi ST. Calcium-induced calcium release from 1987; 67: 386-390. fragmented sarcoplasmic reticulum. Journal of Biochemistry 1979; 86: 1147-1150. 24. Katsuoka M, Kobayashi K, Ohnishi ST. Volatile 12. Ohnishi ST. Calcium-induced calcium release as a gated anesthetics decrease calcium content of isolated myocalcium transport. In: Ohnishi ST, Endo M, eds. The cytes. Anesthesiology 1989; (in press). Mechanism of Gated Calcium Transport Across Biological 25. Lynch C, Vogel S, Sperelakis N. Halothane depression of Membranes. New York: Academic Press, 1981; 275myocardial slow action potentials. Anesthesiology 1981; 294 55: 360-368. 13. Ohnishi ST, Waring AJ, Fang SG, Horiuchi K, Flick JL, 26. Terrar DA, Victory JGG. Effects of halothane on Sadanaga KK, Ohnishi T. Abnormal membrane propermembrane currents associated with contraction in single ties of the sarcoplasmic reticulum of pigs susceptible to myocytes isolated from guinea-pig ventricle. British malignant hyperthermia: Modes of action of halothane, Journal of Pharmacology 1988; 94: 500-508. caffeine, dantrolene and two other drugs. Archives of 27. Price HL, Ohnishi ST. Effects of anesthetics on the heart. Biochemistry and Biophysics 1986; 247: 294-301. Federation Proceedings 1980; 39: 575-1579.
INHALATION ANAESTHETICS DECREASE CALCIUM CONTENT OF CARDIAC SARCOPLASMIC RETICULUMf M. KATSUOKA AND S. T. OHNISHI
MATERIALS AND METHODS
Langendorff perfusion model MASAVUKI KATSUOKA, M.S.; S. TSUYOSHI OHNISHI, PH.D.;
Membrane Research Institute, University City Science Center, 3401 Market Street, Philadelphia, Pa 19104 U.S.A. Accepted for Publication: December 6, 1988. fPreliminary results were presented at the Annual Meeting of the Biophysical Society at Phoenix, Arizona (March, 1988).
Male Sprague-Dawley rats (weight 300-400 g) were anaesthetized by i.p. injection of pentobarbitone 50 mg kg"1. The heart was removed quickly and immersed in ice cold Krebs-Henseleit bicarbonate solution (KHB solution; content
Downloaded from http://bja.oxfordjournals.org/ at University of Bath Library & Learning Centre on June 22, 2015
It is known that general anaesthetics markedly reduce myocardial contractility [1-4]. In in vitro SUMMARY experiments, Price [5,6] found that anaesthetic- In a Langendorff perfused rat heart model, induced reduction in contractile force of papillary caffeine was shown to have a negative inotropic muscle was antagonized by increasing calcium effect similar to that of inhalation anaesthetics. concentration in the bathing medium. One of the Both inhalation anaesthetics and caffeine desites of action suggested by several investigators creased calcium content of left ventricular muscle was the plasma membrane. Langer, Serena and as measured by atomic absorption spectroscope. Nudd [7] suggested that membrane surface- Halothane 2.8% decreased left ventricular pressbound calcium plays a role in the contraction of ure by 75.4 (SEM 4.4)% and decreased the cardiac muscle. Using a La3+ exchange technique, calcium content by 30%. Enflurane 5.1% deOhnishi and colleagues [8] have shown that creased the pressure by 72.4 (6.0) % and dehalothane reduces the amount of La3+-exchange- creased calcium content by 31%. Both halothane able calcium in trabecular muscle in proportion to and enflurane decreased the calcium content of the decrease in contractility. They showed that sarcoplasmic reticulum (SR) as measured by the calcium binding of the preparation from caffeine-induced calcium release. There was a cardiac plasma membrane lipoprotein was in- high degree of correlation between the negative creased in the presence of general anaesthetics and inotropic effect of inhalation anaesthetics and ethanol [9]. the decrease in calcium content of the SR While calcium bound to the plasma membrane (x = 0.95 for halothane, r = 0.91 for enflurane). may play a role in cardiac muscle contraction, the These data suggest that inhalation anaesthetics major site of interest is the sarcoplasmic reticulum increase the calcium permeability of cardiac SR, (SR). Su and Kerrick [10] showed that halothane thereby decreasing the calcium content of SR. caused myocardial depression by inhibiting cal- resulting in the negative inotropic effect. cium uptake of SR in skinned fibres. Ohnishi [11,12] found that both halothane and caffeine increased the calcium-induced calcium release from the SR. Using SR prepared from malignant using a Langendorff perfused rat heart model. hyperthermia-susceptible skeletal muscle, he We measured both the total calcium content of the found that both caffeine and halothane increased left ventricular muscle with atomic absorption the calcium permeability of the SR [13]. We spectroscopy and the amount of calcium ion postulated that halothane may also increase released from the SR using a caffeine-induced calcium permeability of the cardiac SR, thereby calcium release method [14, 15]. decreasing the calcium content of the SR. In the present study, we have tested this hypothesis
BRITISH JOURNAL OF ANAESTHESIA
670 Perfusate •
Anaesth. gas
Ca2*-free' buffer
Ca2+-free buffer + caffeine
Control
o o Enflurane
1min
1s
FIG. 1. Caffeine-induced contraction: Control (no addition); effect of 2.8% halothane and effect of 5.1 % enflurane. The heart was perfused with inhalation anaesthetic (Anaesth.) for a few minutes and with Ca2+-free buffer for 30 s, then with Ca2+-free buffer containing caffeine 40 mmol litre"1.
(mmol litre"1): NaCl 118, KC1 4.7, CaCl2 1.25, MgSO4 1.25, KH2PO4 1.25, NaHCO3 24, glucose 15; pH adjusted to 7.4 when equilibrated with 5 % carbon dioxide in oxygen at 37 °C). Extraneous tissue was removed and the aorta was cannulated on the perfusion apparatus. The left ventricular pressure was measured with a latex balloon connected to a Statham PB 23 transducer via a thin catheter. The balloon was inserted through the mitral valve and filled with water (volume 0.1-0.2 ml).
at 2000 rev min"1 for 10 min and the calcium concentration of the supernatant measured using a polarized Zeeman effect atomic absorption spectrophotometer (Hitachi Z-8000). The pellet was dissolved in 30% potassium hydroxide 10 ml and the protein concentration determined by a biuret method. Measurements were made in triplicates. Estimation of calcium content of sarcoplasmic reticulum using caffeine
Calcium content of SR was measured by the procedures reported by Endo [14,15] and Heide and colleagues [16], with a small modification. The heart was stimulated continuously (5 Hz). The perfused heart was stimulated by electric After equilibration with KHB solution for pulses (10 V, 4-5 Hz, 10 ms duration). The KHB 15 2+min, the perfusing solution was switched to a solution was changed to a KHB solution con- Ca -free KHB2+ solution for 30 s then changed solution containing taining either caffeine or anaesthetic agents (the again, to a Ca -free KHB 1 latter vaporized from a Drager vaporizer and caffeine 40 mmol litre" . Endo found that the bubbled into the solution) and perfused for calcium release at this high concentration of lOmin. The pressure decreased and reached a caffeine represented the amount of calcium left in new plateau value within the first 5 min. The per- the SR [14,15]. Several contractions of the heart fusing solution was changed to a Ca2+-free KHB were observed (fig. 1) and the height of each solution, and perfused for 1 min. The left ven- contraction measured. It was assumed that the tricular muscle was cut into small pieces with height of each contraction was related to calcium scissors and homogenized with a polytron hom- release from the SR and thus the summation of ogenizer (volume 10 ml). The homogenate was each contraction was regarded as the total calcium SR in the presence of caffeine extracted with HC1 2 mol litre"1 for 24 h with release from the 1 constant shaking. The suspension was centrifuged 40 mmol litre" .
Relationship between left ventricular developed pressure (LVDP) and calcium content of the left ventricular muscle
Downloaded from http://bja.oxfordjournals.org/ at University of Bath Library & Learning Centre on June 22, 2015
Halothane
INHALATION ANAESTHETICS AND THE HEART Determination of anaesthetic concentration
671
100-1
Concentrations of halothane and enflurane from the respective vaporizers were determined by gas chromatography. a 504
Data analysis
Each datum point was obtained from six separate experiments. Statistical analysis was performed using Student's t test. RESULTS
2
Negative inotropic effects of caffeine, halothane and enflurane
When the heart was perfused with caffeine, diastolic pressure increased. Systolic pressure increased immediately and transiently, then decreased to a new plateau value which was smaller than the original one (negative inotropic effect). Similar changes were observed with both halothane and enflurane (fig. 2). LVDP was depressed by these agents in a dose-related manner (fig. 3). A reduction of 50% was produced by 1.8% halothane and 4.0 % enflurane.
H
H
Caffeine (mmol litre"1) 4 8
ra
10 Anaesthetic gas (7.) FIG. 4. Effects of caffeine ( # ) , halothane (O) and enflurane (A) on calcium content of left ventricular muscle (mean and SEM) (n = 6). Calcium content was measured by atomic absorption spectrophotometry. Significant difference from normal perfusate: * P < 0.05; ** P < 0.01. Halothane
Caffeine
1
6 10 Anaesthetic gas (%) FIG. 3. Effects of caffeine ( # ) , halothane (O) and enflurane (A) on the developed pressure (LVDP) expressed as a percentage of contraction perfused with buffer solution without agents (mean and SEM) (n = 6).
Effects of caffeine, halothane and enflurane on calcium content of left ventricular muscle
All these agents decreased the calcium content of left ventricular muscle as measured by atomic absorption spectrophotometry (fig. 4). When the heart was perfused with 2.8% halothane, 5.1% enflurane or caffeine 2 mmol litre"1, the calcium content was decreased to 70.3 (9.5) % (P < 0.05), 69.4 (6.9)% (P<0.05) and 56.1 (6.9)%
—I
Enflurane
b>4o
e CD
1% Halothane
u | '_o 03
O
21 Enflurane
1s
1min
FIG. 2. Effects of caffeine 4 mmol litre" 1 , 1 % halothane and 2 % enflurane on the left ventricular developed pressure (LVDP) of the isolated heart. Arrows indicate change in perfusate.
50 LVDPI/J)
100
50 LVDP (7.)
100
FIG. 5. Relationship between left ventricular developed pressure (LVDP) and calcium content in left ventricular muscle: effects of halothane (r = 0.65, P < 0.05) and enflurane (r = 0.60, P < 0.05).
Downloaded from http://bja.oxfordjournals.org/ at University of Bath Library & Learning Centre on June 22, 2015
Caffeine (mmol litre'1) 4 8
0J 0
BRITISH JOURNAL OF ANAESTHESIA
672 100n
Halothane
Enflurane
503 T3
c
•i
0 50
100
50
100
LVDP (7.) LVDPU) a FIG. 6. Effect of halothane (r = 0.95, P< 0.001) and enflurane (r = 0.91, P < 0.001) on the relationship between left ventricular developed pressure (LVDP: percentage of control) and calcium content in the SR. Calcium content in the SR was measured as a caffeine-induced contraction and expressed as a percentage of control.
(P < 0.01) of control, respectively. The relationship between LVDP and the calcium content of the left ventricular muscle was similar with halothane and enflurane (r = 0.65, P < 0.05 and r = 0.60, P < 0.05, respectively) (fig. 5). Relationship between LVDP and calcium content in the SR In this experiment, calcium content was estimated by caffeine-induced calcium release from the SR (fig. 1). There was a correlation between LVDP and calcium content of the SR (r = 0.95, P < 0.001 and r = 0.91, P < 0.001 for halothane and enflurane, respectively) (fig. 6). DISCUSSION
Although inhalation anaesthetics are well known to reduce contractile force of the heart, the exact mechanism of action is not well understood. Using isolated skeletal muscle SR, it has been shown that inhalation anaesthetics, such as halothane and enflurane, stimulate calcium release from the SR [17]. It has also been demonstrated that both caffeine and inhalation anaesthetics increase the calcium permeability of SR membranes [11,12] and it is well known that caffeine causes calcium release from the SR [14-17]. These findings suggest that there are close similarities between the mechanism of action of caffeine and that of inhalation anaesthetics on the SR, and that the mechanism of negative inotropism may be related to the stimulated calcium release from the SR. Using a Langendorff perfused heart model, we attempted to test this hypothesis.
ACKNOWLEDGEMENTS This work was supported by NIH grants GM 33025 and GM 35681. We thank Dr Bryan Marshall, Department of Anesthesiology, University of Pennsylvania, for determining the halothane and enflurane concentrations.
REFERENCES 1. Brewster WR, Isaacs JP, Waing-Anderson T. Depressant effect of ether on myocardium of the dog and its modification by reflex release of epinephrine and norepinephrine. Journal of Pharmacology and Experimental Therapeutics 1953; 175: 399-414.
Downloaded from http://bja.oxfordjournals.org/ at University of Bath Library & Learning Centre on June 22, 2015
CD
First, we demonstrated that caffeine, a well known SR calcium releasing agent [14-17], has a negative inotropic effect on cardiac muscle (figs 2, 3). The action of caffeine and inhalation anaesthetics on the SR seemed to be similar: they caused an immediate increase in systolic pressure followed by a decrease, and they caused an increase in diastolic pressure (fig. 2). Second, we measured the calcium content of the left ventricular muscle using atomic absorption spectroscopy. Both inhalation anaesthetics and caffeine decreased the calcium content in a doserelated manner (fig. 4). Third, we used a method of estimating the calcium content of SR [14-16] and demonstrated that the tension developed in a muscle fibre in the presence of high concentrations of caffeine was related to the amount of calcium released from the SR. We applied the method to the heart by assuming that each contraction represented die amount of calcium released from cardiac SR. Caffeine-induced contraction of the heart was decreased with an increase in concentration of inhalation anaesthetic (fig. 1), suggesting, in agreement with results obtained by others [18,19], that inhalation anaesthetics decrease the calcium content of the SR. Several mechanisms may be involved in the negative inotropic effect of inhalation anaesthetics. They affect calcium binding of the plasma membrane [8,9,20] and the SR [10,18,19,2124], and the action potential [25,26]. Our data suggest the possibility of a new mechanism involving the myocardial SR. As postulated previously [27], inhalation anaesthetics increase the calcium permeability of the cardiac SR (and of skeletal SR). This may cause a decrease in calcium content of the SR resulting in a decrease in the maximum developed tension of heart muscle,— that is, negative inotropism.
INHALATION ANAESTHETICS AND THE HEART
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