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Effect of luteinizing hormone-releasing hormone on the mechanical activity of the guinea-pig cardiac muscle
Gen. Pharmac. Vol. 20, No. 6, pp. 743-746, 1989 Printed in Great Britain.All rights reserved
0306-3623/89$3.00+ 0.00 Copyright © 1989PergamonPress plc
EFFECT OF LUTEINIZING HORMONE-RELEASING HORMONE ON THE MECHANICAL ACTIVITY OF THE GUINEA-PIG CARDIAC MUSCLE JUNICHI HASEGAWA,MAKOTOSAITOH,SHOZOHIRAI, MASAHARUFUKUKI, HIROSHI KOTAKE,AKIOYOSHIDA,CHIAKISHIGEMASAand HIROTOMASHIBA Department of Internal Medicine, Tottori University School of Medicine, Yonago 683, Japan. (Tel. 0859-33-111l; Fax 0859-22-7239) (Received 10 January 1989) Abstract--1. The inotropic effect of physiological concentrations of luteinizing hormone-releasing hormone (LH-RH) in isolated guinea-pig cardiac muscles was studied. 2. LH-RH increased the contractile force elicited by either fast and slow responses in a dose-dependent manner. 3. These effects of LH-RH were affected by phentolamine and diltiazem but not by propranolol and cold condition. 4. This study showed the positive inotropic effect of LH-RH on myocardium through the affection to intracellular Ca, and the difference from the effect of cardiotonic steroids.
INTRODUCTION
Luteinizing hormone-releasing hormone (LH-RH) was first characterized as a hypothalamic hormone capable of stimulating the hypophyseal secretion of luteinizing and follicle-stimulatinghormones in mammals (Schally et al., 1971). Recently, L H - R H has been shown to be present in various regions of the body and to have various effects, including the interaction with the mammalian sympathetic nervous system (Wilson et al., 1984). Furthermore, LH-RH-like peptide has been reported to function as a peptidergic transmitter in sympathetic neurons (Emson, 1979; Htkfelt et al., 1980; Jan et al., 1980; Snyder, 1980; Jan and Jan, 1982; Katayama and Nishi, 1982). As for the cardiac effects of peptide hormone, L H - R H was shown to decrease the activity of Na, K-ATPase (Isachenkov et al., 1979; Kravtsov and Isachenkov, 1979) and the frequency of contraction (Kravtsov et al., 1979) of the rat heart. On the other hand, other hypothalamic hormones such as thyrotropin releasing hormone and growth hormone releasing factor have been suggested to have the direct inotropic effect through an increase in the slow inward Ca current (Hasegawa et al., 1988a, b). It is not known whether L H - R H affects the contractile force of cardiac muscle without the sympathetic interaction and if it does, whether inhibition of Na, K-ATPase activity is the mechanism underlying the inotropic effect of LH-RH. We examined the influence of L H - R H on the mechanical activity of isolated guinea-pig ventricular muscle, to investigate the physiological significance of L H - R H on cardiac function.
0.8-1.2ram in diameter) were rapidly exised from the right ventricle and mounted on an organ bath perfused with oxygenated (95% 02, 5% CO2) Tyrode solution at 36.0 _+0.3°C. The flow rate was controlled at 3 ml/min using a micro-tube pump (Tokyo Rikakikai, MP-3). The basal part of the muscle was pinned to the silicon rubber floor of the bath by means of two stainless-steel pins connected to a stimulator (Nihon Kohden, SEN-3201) through an isolator (Nihon Kohden, SS-102J). The rate of stimulation was l Hz, the duration of the pulses 2 msec and the voltage 1.5 times the threshold. For measurement of the force of contraction, the chordal end of the muscle was connected by means of a short silk thread to the hook of a force displacement transducer (Nihon Kohden, TB-612T). Contractile forces were monitored on an oscilloscope (Nihon Kohden, VC-10), photographed by a camera (Nihon Kohden RLG-6201) and recorded simultaneously on a recticorder (Nihon Kohden, RJG-4122). Normal Tyrode solution had the followingcomposition in raM: NaC1 132, KC1 4, NaHCO 3 12, NaH2PO4 0.4, CaC12 1.8, MgC12 1 and glucose 10. The high K solution was prepared by increasing the KC1 concentration to 27 mM and by adding 0.2 mM BaC12 to elicit the Ca-dependent slow response without application of catecholamine (Ehara and Inazawa, 1980). The following compounds were added to the solution just before using: LH-RH (L-Pyroglutamyl-L-histidyl-Ltryptophyl- L-seryl-L-tyrosyl- glycyl-L-leucyl-L arginyl- Lprolyl-glycinamide diacetate) (Tanabe), DL-propranolol (Sumitomo), phentolamine (Ciba-Geigy), and diltiazem (Tanabe). To avoid interference of endogeneous noradrenaline, the catecholamine contents of hearts were depleted by pretreating the guinea-pigs subcutaneously with 2.5 ~ 5 mg/kg reserpine one day before they were killed.
MATERIALS AND METHODS
Guinea-pigs (200-350 g) were killed by a blow on the head, and the papillary muscles (2.5-5mm long and Address correspondence to: Dr Junichi Hasegawa, Department of Internal Medicine, Tottori University School of Medicine, 36-1 Nishimachi, Yonago 683, Japan. 743
-
RESULTS L H - R H increased the force of contraction of the guinea-pig cardiac muscle. The inotropic effect of L H - R H persisted for over 30 min without any transient decreases (Fig. 1A). Occasionally it caused automatic activities even in ventricular muscles. In contrast to the finding that 10-~°M L H - R H did
744
JUNICHI HASEGAWA et al.
A
IIIIlli~~li.... o
] 5gg .
.
LH-RH
100 mg
B
]%
c
2 mln Fig. 1. Effect of LH-RH on contractile force. (A) Low speed record of a sustained inotropic response to 10 -7 M LH-RH exposure in high K solution. In this and subsequent figures, the time period of drug application is indicated by the horizontal bar under the force strip. (B) 10-~0M LH-RH failed to change the contractile force of the muscle, and 10-7 M LH-RH elicited automatic contractions which had a higher rate and a greater force. The arrow under the force strip indicates the point of an application of l0 "-7 M LH-RH. High speed records of the contractile force of the muscle are shown in the control, during automatic responses triggered by exposure to 10 -7 M LH-RH and in the recovery stages. (C) The responses in contractile force to the 10-8 M (left) and 10 -7 M (right) LH-RH in normal Tyrode solution.
not elicit any change in contractile force, 10 -7 M L H - R H induced automatic contractions (Fig. 1B). For another preparation, the dose-dependency of the inotropic action of L H - R H is shown in Fig. IC. Dose-response relationship of L H - R H in 6 experiments examined in the method of repetitive trials similar to Fig. 1C are shown in Fig. 2. Many hormones and chemicals enhance the physiological activities of the myocardium through specific receptors. In Figs 3 and 4, the effects of pretreatment with 1 # M phentolamine (Fig. 3B) and 1/~M propranolol (second trace in Fig. 4B and Fig. 4C) were tested as antagonists to alpha- and beta-adrenoceptors, respectively. It is apparent that the inotropic effect of L H - R H was reduced by phenatolamine, but was not affected by propranolol. The involvement of the slow Ca channel in the
tO ¢1
inotropism of L H - R H was also examined (Fig. 4). Pretreatment with diltiazem (10-6M) reduced the effect of L H - R H on contractile force (Fig. 4A). Although 1/~M diltiazem did not reduce the inotropic effect of L H - R H in normal Tyrode solution when applied during L H - R H treatment (second trace in Fig. 4B), it did strongly suppress the contractility in the high K solution (Fig. 4C). A high dose of diltiazem (10 -4 M) was needed to suppress the contractility enhanced by L H - R H in normal Tyrode solution (not shown). The inotropic effect of L H - R H on the cardiac muscle was observed even at a low temperature (21°C) (Fig. 5), in contrast to the inotropic effect of 10 -7 M strophanthidin which enhanced the contraction by 80% at 36°C and failed to affect it at 21°C (not shown).
I A
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.....
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....
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LH-RH concentration (-log M) Fig. 2. Dose-response relationship for LH-RH. Dose dependency of the inotropic effect of LH-RH are shown. Contractile force of muscles are expressed as % of control value in the ordinate (mean + SE), (n = 4-6).
I
I
2 min Fig. 3. Effect of phentolamine on the inotropic effect of LH-RH. (A) Control contractile force response of muscle exposed to 10-TM LH-RH in Tyrode solution, (B) Response to 10 -7 M LH-RH in the presence of 10-6 M phentolamine, (C) Response to 10-TM LH-RH in the recovery stage.
Inotropic effect of LH-RH
DII
s2 min ~ Fig. 4. Effects of diltiazem and propranolol on the inotropic
effect of LH-RH. (A) Control response to 10-7 M LH-RH (upper), and the response in the presence of 10-SM diltiazem (lower strip). (B) Control response to 10-6M LH-RH (top strip). Contractile force responses to the application of LH-RH and LH-RH plus 10-6 M diltiazem in the presence of 10-6M propranolol (middle strip). Responses to 10-7 M LH-RH and LH-RH plus 5' 10-6 M diltiazem in high K solution (bottom strip).
DISCUSSION
L H - R H has been shown to increase blood pressure in toads by mediating the increase in catecholamine concentration (Wilson et al., 1984). The same group also suggested that L H - R H increased the concentration of norepinephrine and epinephrine which in turn affected the blood pressure. The Co-existence and co-release of cholinergic transmitters and L H - R H like peptide in sympathetic ganglia suggested another role of L H - R H in the interaction with classical transmitters (Jan and Jan, 1983). On the other hand, L H - R H has been reported to have a cardiotonic effect by decreasing Na, K-ATPase activity mediating its specific receptor on the cardiac cell membrane (Isachenkov et al., 1979). L H - R H was shown to have a positive inotropic effect on guinea-pig cardiac muscle in the present study. Since the preparations
]so
im JJl!1
I
I
1 mg
2 rain Fig. 5. Effect of low temperature on the inotropic action of LH-RH. The upper strip shows the control response to 10-7 M LH-RH exposure at 36°C. The lower strip shows the increase in contractile force of muscle during 10-7 M LH-RH exposure even at 21°C. The frequency of stimulation was reduced from I-0.2 Hz.
745
used in the present study were independent of the sympathetic nervous system and intrinsic catecholamines, a direct effect on the cardiac muscle cells seems to be the mechanism responsible for the inotropic effect of L H - R H . The possibility that an interaction exists between the alpha-adrenergic and L H - R H mediating systems in myocardium is proposed to explain the finding that the inotropic effect of L H - R H was suppressed by the application of phentolamine but not propranolol. Recently, some hypothalamic hormones have been shown to have the inotropic effect on the guinea-pig ventricular myocardium. They suggest the increase in intracellular Ca concentration through an increase in the slow inward Ca currents of the muscle as the mechanism underlying the inotropic effect of these hormones. This concept seems to be possible to apply to the effect of L H - R H from the fact that the inotropic effect of L H - R H was observed in the high K solution in which the slow inward Ca current had the major role in the generation of contraction (Wiggins, 1981). The inotropic effect of L H - R H was present even under cold conditions in which Na, K-ATPase activity was supposedly suppressed enough (LaManna and Ferrier, 1981) and further inhibition of Na, K-ATPase induced by strophanthidin failed to affect the contractility. Our results provide an evidence that the LH-RH-induced enhancement of cardiac contractile force is not mediated only by an inhibition of the Na, K-ATPase activity. Diltiazem decreased the inotropic effect of L H - R H in the high K solution in which muscle contraction is mediated mainly by the slow inward current of the membrane (Wiggins, 1981), but had less of an effect in normal Tyrode solution. This suggests the importance of the intracellular stored Ca, as well as Ca intrusion across the cell membrane, in manifesting the effects of L H - R H . Since it is known that the effects of cardiotonic steroids on contractions or 4SCa uptake in cardiac muscle is relatively insensitive to organic Ca antagonists (McCans et al., 1974; Watanabe and Besch, 1974; Burt and Laner, 1982) and that cardiotonic steroids did not exert their inotropic actions in K-depolarized papillary muscle (Thyrum, 1974; Watanabe and Besch, 1974), our data suggest that the inotropic effect of L H - R H differs from those of cardiotonic steroids. CONCLUSION
These results suggest that L H - R H has a positive inotropic effect in the guinea-pig ventricular myocardium, most likely by affecting the calcium handling system of the cell in a different manner to that of cardiotonic steroids. SUMMARY
The effect of 1 0 - ] 2 - 1 0 -6 M luteinizing hormonereleasing hormone ( L H - R H ) on the mechanical activity of isolated guinea-pig cardiac muscle pretreated with reserpine was studied using a force transducer. L H - R H increased the contractile force of muscles in normal Tyrode and in high K (27 raM) solution in a dose-dependent manner and occasionally elicited automatic activities. The inotropic effect
746
JUNICHI HASEGAWAet al.
of L H - R H was suppressed by phentolamine but was not affected by propranolol. Diltiazem suppressed contractions enhanced by L H - R H in the high K solution and in normal Tyrode. L H - R H was found to exert its inotropic effects even at low temperature in which Na, K - A T P a s e activity was suppressed enough. It is suggested that L H - R H has a positive inotropic effect on guinea-pig ventricular myocardium, most likely by affecting intracellular Ca, and that the effect is different from cardiotonic steroids. REFERENCES
Burt J. M. and Langer G. A. (1982) Ca ++ distribution after Na ÷ pump inhibition in cultured neonatal rat myocardial cells. Circ. Res. 51, 543-550. Ehara T. and Inazawa M. (1980) Calcium-dependent slow action potentials in potassium-depolarized guinea-pig ventricular myocardium enhanced by barium ions. Naunyn Schmiedeberg's Archs Pharmac. 315, 47-54. Emson P. (1979) Peptides as neurotransmitter candidates in the mammalian CNS. Progr. Neurobiol. 13, 61-116. Hasegawa J., Hirai S., Kotake H., Hisatome I. and Mashiba H. (1988a) Inotropic effect of thyrotropin releasing hormone on the guinea-pig myocardium. Endocrinology 123, 2805-281 I. Hasegawa J., Saitoh M., Hirai S., Kinugawa T., Kotake H. and Mashiba H. (1988b) Positive inotropic effect of growth hormone releasing factor in the guinea-pig cardiac muscles in vitro. Horm. Metab. Res. 20, 130-131. H6kfelt T., Johansson O., Ljungdahl A., Lundberg J. M. and Schultzber M. (1980) Peptidergic neurones. Nature 284, 515-521. Isachenkov V. A., Bakalkin G. Ya., Kravtsov G. M., Komissarova E. N. and Pokudin N. I. (1979) Cardiotropic effects of Luliberin. Interaction with rat heart sarcolemmal membrane. Biokhimiia 44, 387-393. Jan L. Y. and Jan Y. N. (1982) Peptidergic transmission in sympathetic ganglia of the frog. J. Physiol., Lond. 327, 219-246. Jan Y. N. and Jan L. Y. (1983) Coexistence and
corelease of cholinergic and peptidergic transmitters in frog sympathetic ganglia. Fedn Proc. 42, 2929-2933. Jan Y. N., Jan L. Y. and Kuffler S. (1980) Further evidence for peptidergic transmission in sympathetic ganglia. Proc. natn. Acad. Sci., U.S.A. 77(8), 5008-5012. Katayama Y. and Nishi S. (1982) Voltage-clamp analysis of peptidergic slow depolarization in bullfrog sympathetic ganglion cells. J. Physiol., Lond., 333, 305-313. Kravtsov G. M. and Isachenkov V. A. (1979) Effect of luliberine on the activity of Na, K-ATPase and Ca-ATPase of the rat heart sarcolemma. Biull. Eksp. biol. Med. 87, 222-224. Kravtsov G. M., Isachenkov V. A. and Evteeva T. V. (1979) Effects of Hypothalamic LH-RH (Lulebirine) on the electrical activity of the rat isolated heart. Fiziol. Zh. S.S.S.R. 65, 133-137. LaManna V. R. and Ferrier G. R. (1981) Electrophysiological effects of insuline on normal and depressed cardiac tissues. Am. J. Physiol. 240, H636-H644. McCans J. L., Lindenmayer G. E., Munson R. G., Evans R. W. and Schwartz A. (1974) A dissociation of positive staircase (Bowditch) from ouabain-induced positive inotropism. Circ. Res. 35, 439-447. Schally A. V., Arimura A., Kastin A. J., Matsuo H., Baba Y., Redding T. W,, Nair R. M. G., Debeljuk L., White W. F. (1971) Gonadotropin-releasing hormone: one polypeptide regulates secretion of luteinizing and follicle-stimulating hormones. Science 173, 1036-1038. Snyder S. H. (1980) Brain peptides as neurotransmitters. Science 209, 976-983. Thyrum P. T. (1974) Inotropic stimuli and systolic transmembrane calcium flow in depolarized guinea-pig atria. J. Pharmac. Exp. Ther. 188, 166-179. Watanabe A. M. and Besch H. R. Jr. (1974) Cyclic adenosine monophosphate modulation of slow calcium influx channels in guinea pig hearts. Circ. Res. 35, 316-324. Wiggins J. R. (1981) Inotropic actions of isoproterenol in cat ventricular muscle. Effects of extracellular potassium. Circ. Res. 49, 718-725. Wilson J. X., Van Vliet B. N. and West N. H. (1984) Gonadotropin-releasing hormone increase plasma catecholamines and blood pressure in toads. Neuroendocrinology 39, 437-441.
0306-3623/89$3.00+ 0.00 Copyright © 1989PergamonPress plc
EFFECT OF LUTEINIZING HORMONE-RELEASING HORMONE ON THE MECHANICAL ACTIVITY OF THE GUINEA-PIG CARDIAC MUSCLE JUNICHI HASEGAWA,MAKOTOSAITOH,SHOZOHIRAI, MASAHARUFUKUKI, HIROSHI KOTAKE,AKIOYOSHIDA,CHIAKISHIGEMASAand HIROTOMASHIBA Department of Internal Medicine, Tottori University School of Medicine, Yonago 683, Japan. (Tel. 0859-33-111l; Fax 0859-22-7239) (Received 10 January 1989) Abstract--1. The inotropic effect of physiological concentrations of luteinizing hormone-releasing hormone (LH-RH) in isolated guinea-pig cardiac muscles was studied. 2. LH-RH increased the contractile force elicited by either fast and slow responses in a dose-dependent manner. 3. These effects of LH-RH were affected by phentolamine and diltiazem but not by propranolol and cold condition. 4. This study showed the positive inotropic effect of LH-RH on myocardium through the affection to intracellular Ca, and the difference from the effect of cardiotonic steroids.
INTRODUCTION
Luteinizing hormone-releasing hormone (LH-RH) was first characterized as a hypothalamic hormone capable of stimulating the hypophyseal secretion of luteinizing and follicle-stimulatinghormones in mammals (Schally et al., 1971). Recently, L H - R H has been shown to be present in various regions of the body and to have various effects, including the interaction with the mammalian sympathetic nervous system (Wilson et al., 1984). Furthermore, LH-RH-like peptide has been reported to function as a peptidergic transmitter in sympathetic neurons (Emson, 1979; Htkfelt et al., 1980; Jan et al., 1980; Snyder, 1980; Jan and Jan, 1982; Katayama and Nishi, 1982). As for the cardiac effects of peptide hormone, L H - R H was shown to decrease the activity of Na, K-ATPase (Isachenkov et al., 1979; Kravtsov and Isachenkov, 1979) and the frequency of contraction (Kravtsov et al., 1979) of the rat heart. On the other hand, other hypothalamic hormones such as thyrotropin releasing hormone and growth hormone releasing factor have been suggested to have the direct inotropic effect through an increase in the slow inward Ca current (Hasegawa et al., 1988a, b). It is not known whether L H - R H affects the contractile force of cardiac muscle without the sympathetic interaction and if it does, whether inhibition of Na, K-ATPase activity is the mechanism underlying the inotropic effect of LH-RH. We examined the influence of L H - R H on the mechanical activity of isolated guinea-pig ventricular muscle, to investigate the physiological significance of L H - R H on cardiac function.
0.8-1.2ram in diameter) were rapidly exised from the right ventricle and mounted on an organ bath perfused with oxygenated (95% 02, 5% CO2) Tyrode solution at 36.0 _+0.3°C. The flow rate was controlled at 3 ml/min using a micro-tube pump (Tokyo Rikakikai, MP-3). The basal part of the muscle was pinned to the silicon rubber floor of the bath by means of two stainless-steel pins connected to a stimulator (Nihon Kohden, SEN-3201) through an isolator (Nihon Kohden, SS-102J). The rate of stimulation was l Hz, the duration of the pulses 2 msec and the voltage 1.5 times the threshold. For measurement of the force of contraction, the chordal end of the muscle was connected by means of a short silk thread to the hook of a force displacement transducer (Nihon Kohden, TB-612T). Contractile forces were monitored on an oscilloscope (Nihon Kohden, VC-10), photographed by a camera (Nihon Kohden RLG-6201) and recorded simultaneously on a recticorder (Nihon Kohden, RJG-4122). Normal Tyrode solution had the followingcomposition in raM: NaC1 132, KC1 4, NaHCO 3 12, NaH2PO4 0.4, CaC12 1.8, MgC12 1 and glucose 10. The high K solution was prepared by increasing the KC1 concentration to 27 mM and by adding 0.2 mM BaC12 to elicit the Ca-dependent slow response without application of catecholamine (Ehara and Inazawa, 1980). The following compounds were added to the solution just before using: LH-RH (L-Pyroglutamyl-L-histidyl-Ltryptophyl- L-seryl-L-tyrosyl- glycyl-L-leucyl-L arginyl- Lprolyl-glycinamide diacetate) (Tanabe), DL-propranolol (Sumitomo), phentolamine (Ciba-Geigy), and diltiazem (Tanabe). To avoid interference of endogeneous noradrenaline, the catecholamine contents of hearts were depleted by pretreating the guinea-pigs subcutaneously with 2.5 ~ 5 mg/kg reserpine one day before they were killed.
MATERIALS AND METHODS
Guinea-pigs (200-350 g) were killed by a blow on the head, and the papillary muscles (2.5-5mm long and Address correspondence to: Dr Junichi Hasegawa, Department of Internal Medicine, Tottori University School of Medicine, 36-1 Nishimachi, Yonago 683, Japan. 743
-
RESULTS L H - R H increased the force of contraction of the guinea-pig cardiac muscle. The inotropic effect of L H - R H persisted for over 30 min without any transient decreases (Fig. 1A). Occasionally it caused automatic activities even in ventricular muscles. In contrast to the finding that 10-~°M L H - R H did
744
JUNICHI HASEGAWA et al.
A
IIIIlli~~li.... o
] 5gg .
.
LH-RH
100 mg
B
]%
c
2 mln Fig. 1. Effect of LH-RH on contractile force. (A) Low speed record of a sustained inotropic response to 10 -7 M LH-RH exposure in high K solution. In this and subsequent figures, the time period of drug application is indicated by the horizontal bar under the force strip. (B) 10-~0M LH-RH failed to change the contractile force of the muscle, and 10-7 M LH-RH elicited automatic contractions which had a higher rate and a greater force. The arrow under the force strip indicates the point of an application of l0 "-7 M LH-RH. High speed records of the contractile force of the muscle are shown in the control, during automatic responses triggered by exposure to 10 -7 M LH-RH and in the recovery stages. (C) The responses in contractile force to the 10-8 M (left) and 10 -7 M (right) LH-RH in normal Tyrode solution.
not elicit any change in contractile force, 10 -7 M L H - R H induced automatic contractions (Fig. 1B). For another preparation, the dose-dependency of the inotropic action of L H - R H is shown in Fig. IC. Dose-response relationship of L H - R H in 6 experiments examined in the method of repetitive trials similar to Fig. 1C are shown in Fig. 2. Many hormones and chemicals enhance the physiological activities of the myocardium through specific receptors. In Figs 3 and 4, the effects of pretreatment with 1 # M phentolamine (Fig. 3B) and 1/~M propranolol (second trace in Fig. 4B and Fig. 4C) were tested as antagonists to alpha- and beta-adrenoceptors, respectively. It is apparent that the inotropic effect of L H - R H was reduced by phenatolamine, but was not affected by propranolol. The involvement of the slow Ca channel in the
tO ¢1
inotropism of L H - R H was also examined (Fig. 4). Pretreatment with diltiazem (10-6M) reduced the effect of L H - R H on contractile force (Fig. 4A). Although 1/~M diltiazem did not reduce the inotropic effect of L H - R H in normal Tyrode solution when applied during L H - R H treatment (second trace in Fig. 4B), it did strongly suppress the contractility in the high K solution (Fig. 4C). A high dose of diltiazem (10 -4 M) was needed to suppress the contractility enhanced by L H - R H in normal Tyrode solution (not shown). The inotropic effect of L H - R H on the cardiac muscle was observed even at a low temperature (21°C) (Fig. 5), in contrast to the inotropic effect of 10 -7 M strophanthidin which enhanced the contraction by 80% at 36°C and failed to affect it at 21°C (not shown).
I A
200
@
P
o
150
B
.....
~
-" _
....
IIIIII
t
0
c 100
1'2
1'1
1'0
6
8
"l
LH-RH concentration (-log M) Fig. 2. Dose-response relationship for LH-RH. Dose dependency of the inotropic effect of LH-RH are shown. Contractile force of muscles are expressed as % of control value in the ordinate (mean + SE), (n = 4-6).
I
I
2 min Fig. 3. Effect of phentolamine on the inotropic effect of LH-RH. (A) Control contractile force response of muscle exposed to 10-TM LH-RH in Tyrode solution, (B) Response to 10 -7 M LH-RH in the presence of 10-6 M phentolamine, (C) Response to 10-TM LH-RH in the recovery stage.
Inotropic effect of LH-RH
DII
s2 min ~ Fig. 4. Effects of diltiazem and propranolol on the inotropic
effect of LH-RH. (A) Control response to 10-7 M LH-RH (upper), and the response in the presence of 10-SM diltiazem (lower strip). (B) Control response to 10-6M LH-RH (top strip). Contractile force responses to the application of LH-RH and LH-RH plus 10-6 M diltiazem in the presence of 10-6M propranolol (middle strip). Responses to 10-7 M LH-RH and LH-RH plus 5' 10-6 M diltiazem in high K solution (bottom strip).
DISCUSSION
L H - R H has been shown to increase blood pressure in toads by mediating the increase in catecholamine concentration (Wilson et al., 1984). The same group also suggested that L H - R H increased the concentration of norepinephrine and epinephrine which in turn affected the blood pressure. The Co-existence and co-release of cholinergic transmitters and L H - R H like peptide in sympathetic ganglia suggested another role of L H - R H in the interaction with classical transmitters (Jan and Jan, 1983). On the other hand, L H - R H has been reported to have a cardiotonic effect by decreasing Na, K-ATPase activity mediating its specific receptor on the cardiac cell membrane (Isachenkov et al., 1979). L H - R H was shown to have a positive inotropic effect on guinea-pig cardiac muscle in the present study. Since the preparations
]so
im JJl!1
I
I
1 mg
2 rain Fig. 5. Effect of low temperature on the inotropic action of LH-RH. The upper strip shows the control response to 10-7 M LH-RH exposure at 36°C. The lower strip shows the increase in contractile force of muscle during 10-7 M LH-RH exposure even at 21°C. The frequency of stimulation was reduced from I-0.2 Hz.
745
used in the present study were independent of the sympathetic nervous system and intrinsic catecholamines, a direct effect on the cardiac muscle cells seems to be the mechanism responsible for the inotropic effect of L H - R H . The possibility that an interaction exists between the alpha-adrenergic and L H - R H mediating systems in myocardium is proposed to explain the finding that the inotropic effect of L H - R H was suppressed by the application of phentolamine but not propranolol. Recently, some hypothalamic hormones have been shown to have the inotropic effect on the guinea-pig ventricular myocardium. They suggest the increase in intracellular Ca concentration through an increase in the slow inward Ca currents of the muscle as the mechanism underlying the inotropic effect of these hormones. This concept seems to be possible to apply to the effect of L H - R H from the fact that the inotropic effect of L H - R H was observed in the high K solution in which the slow inward Ca current had the major role in the generation of contraction (Wiggins, 1981). The inotropic effect of L H - R H was present even under cold conditions in which Na, K-ATPase activity was supposedly suppressed enough (LaManna and Ferrier, 1981) and further inhibition of Na, K-ATPase induced by strophanthidin failed to affect the contractility. Our results provide an evidence that the LH-RH-induced enhancement of cardiac contractile force is not mediated only by an inhibition of the Na, K-ATPase activity. Diltiazem decreased the inotropic effect of L H - R H in the high K solution in which muscle contraction is mediated mainly by the slow inward current of the membrane (Wiggins, 1981), but had less of an effect in normal Tyrode solution. This suggests the importance of the intracellular stored Ca, as well as Ca intrusion across the cell membrane, in manifesting the effects of L H - R H . Since it is known that the effects of cardiotonic steroids on contractions or 4SCa uptake in cardiac muscle is relatively insensitive to organic Ca antagonists (McCans et al., 1974; Watanabe and Besch, 1974; Burt and Laner, 1982) and that cardiotonic steroids did not exert their inotropic actions in K-depolarized papillary muscle (Thyrum, 1974; Watanabe and Besch, 1974), our data suggest that the inotropic effect of L H - R H differs from those of cardiotonic steroids. CONCLUSION
These results suggest that L H - R H has a positive inotropic effect in the guinea-pig ventricular myocardium, most likely by affecting the calcium handling system of the cell in a different manner to that of cardiotonic steroids. SUMMARY
The effect of 1 0 - ] 2 - 1 0 -6 M luteinizing hormonereleasing hormone ( L H - R H ) on the mechanical activity of isolated guinea-pig cardiac muscle pretreated with reserpine was studied using a force transducer. L H - R H increased the contractile force of muscles in normal Tyrode and in high K (27 raM) solution in a dose-dependent manner and occasionally elicited automatic activities. The inotropic effect
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of L H - R H was suppressed by phentolamine but was not affected by propranolol. Diltiazem suppressed contractions enhanced by L H - R H in the high K solution and in normal Tyrode. L H - R H was found to exert its inotropic effects even at low temperature in which Na, K - A T P a s e activity was suppressed enough. It is suggested that L H - R H has a positive inotropic effect on guinea-pig ventricular myocardium, most likely by affecting intracellular Ca, and that the effect is different from cardiotonic steroids. REFERENCES
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