Seasonal and Sexual Variations in the Responsiveness of Rabbit Hearts to Prolactin B. A. NASSAR, D. F. HORROBIN, M. TYNAN, M. S. MANKU, AND PATRICIA A. DA VIES Department of Physiology, University of Newcastle Medical School, Newcastle upon Tyne NE1 7RU, England prolactin were seen in 5 adult males pretreated with reserpine. Propanolol consistently reversed both the inotropic and chronotropic actions of prolactin. The original experiments were performed in January and February. When tested in May, adult males failed to respond to prolactin and this situation persisted until October when responsiveness again appeared. The same prolactin preparation and procedures were used throughout indicating that the changes must have been due to seasonal variations in the cardiac responsiveness to the hormone. (Endocrinology 97: 1008, 1975)
ABSTRACT. Rabbit hearts perfused by the Langendorff technique were studied. The addition of ovine prolactin (NIH-P-S-10) to the perfusate in a concentration of 50 ng/ml produced rapid increases in both the amplitude and rate of contraction in 33 adult male hearts studied in winter. Prepubertal male animals showed no response, and only 1 out of 12 adult females responded. Pretreatment for 10 days with 2.5 mg/day testosterone propionate led to minimal inotropic but not chronotropic responses in 2 out of 4 prepubertal males and 2 out of 4 adult females to prolactin. Clear responses to
S
EXUAL, seasonal, and temporal variations in responsiveness to hormones have recently attracted attention (1-4). We report here sexual and seasonal variations in the responses of the heart to prolactin. These experimental findings and the principles they illustrate are likely to be of considerable significance in the design and interpretation of studies of both animals and man. Prolactin seems to be in large part responsible for the increase in cardiac output associated with lactation (5-7). This does not prove that prolactin can have a direct cardiac effect since changes in the peripheral circulation will also produce changes in cardiac output (8). In isolated preparations of male rat superior mesenteric vascular beds, ovine prolactin in a concentration of 50 ng/ml potentiated arteriolar responses to noradrenaline and angiotensin without itself causing vasoconstriction (9). Concentrations of 200 ng/ml or over, in contrast, inhibited the effects of the vasoconstrictor agents. Depending on the selectivity of these actions both with respect to the vascular bed concerned and to the relative effects on resistance and capacitance vessels, it is Received March 17, 1975.
possible to conceive of mechanisms whereby either concentration of prolactin could increase venous return and enhance cardiac output. In isolated male rat hearts 50 ng/ml ovine prolactin increased the heart rate while 200 ng/ml slowed it down: both concentrations produced dysrhythmias (10). This paper demonstrates that 50 ng/ml ovine prolactin has inotropic and chronotropic actions on adult male rabbit hearts in winter but not in summer and that female and prepubertal male hearts are unresponsive to the hormone throughout the year. Methods and Materials Rabbits were killed by a blow on the head and the hearts were rapidly removed and perfused with oxygenated Ringer-Locke solution using the Langendorff technique (11,12). (This involves retrograde cannulation of the ascending aorta: the retrograde movement of the perfusate closes the aortic valves, and the fluid passes into the coronary arteries: no circulation passes through the chambers of the hearts (Fig. 1).) Contractions were recorded by means of a hook through the tip of the heart connected to an isometric force transducer. The amplitude and frequency (using an instantaneous rate meter) of the contractions were monitored using a moving paper recorder. After being attached to the perfusion system, the heart was allowed to
1008
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1009
PROLACTIN AND THE HEART stabilise for a 20 min period: hearts which showed dysrhythmias or which failed to achieve a stable rate and amplitude (no more than ±10% variation in eitlier parameter during the last 10 min of the period) were discarded without further testing. After this initial control period the perfusate was then changed from plain Ringer-Locke to a similar solution containing 50 ng/ml ovine prolactin (NIH-P-S-10). If there was no response to the prolactin the experiment was discontinued after 10 min. If there was a response, 3 min after switching to the prolactin solution a single injection of 50/x.g propanol ("Inderal," IC1) was injected into the perfusing cannula. The manufacturer's maximum recommended iv dose in humans is 10 mg: if cardiac plasma output of the human is 2500 ml/min this represents 400 /xg per 100 ml/min. The flow through our rabbit hearts was usually in the region of 30-35 ml/min giving a propanolol dose between 120-140 fxg per 100 ml/min. After a further 3 min a further 20 /xg of propanol was injected. Since prolactin is prepared from tissue which may contain vasoactive peptides and amines such as epinephrine, norepinephrine, dopamine, vasopressin, and oxytocin it is important to consider the possibility that the hormone could have been contaminated with these substances. The superior mesenteric artery of the rat is exquisitely sensitive to the vasoconstrictor actions of epinephrine, norepinephrine, and vasopressin: concentrations of the prolactin up to 5 fxg/m\ failed to have any even transient pressor effect in this preparation, and we therefore consider it extremely unlikely that the cardiac effects of 50 ng/ml prolactin could have been explained by one of these 3 substances. We also tested the direct effects of 5 ng/ml dopamine and oxytocin in the hearts on the assumption that a greater than 10% by weight contamination of the prolactin by such small molecular weight substances would be highly improbable given the methods used for the purification of protein hormones of mol wt about 20,000. Oxytocin in this concentration had no effect. Dopamine produced very small (less than 10%) increases in heart rate and contraction amplitude. However the effects of dopamine like those of epinephrine and norepinephrine occurred equally on male and female hearts. In the past 5 years, this department, in the course of student classes, has tested catecholamine
Perfusion
Cannula
Ascending aorta
Closed aortic valve
Coronary artery
Left ventricle
FIG. 1. Schematic diagram of LangendorfFs technique of cardiac perfusion. The perfusion fluid enters the aorta retrogradely and then, because of the closure of the aortic valves, passes along the coronary arteries. No fluid is pumped through the chambers in the usual way.
effects on about 200 male and female rabbits; no sex differences have been noted. The following Dutch rabbits were used in January and February: a) Eight adult males (at least 3 months post-pubertal) in preliminary experiments during which an appropriate protocol was established, b) 12 adult males in definitive experiments conducted as described, c) 5 adult males treated twice daily for 3 days with 0.5 mg reserpine subcutaneously, d) 4 untreated prepubertal males, e) 4 prepubertal males treated daily for 10 days with 2.5 mg testosterone propionate given subcutaneously in oil, f) 12 untreated adult females, and g) 4 adult females treated daily for 10 days with 2.5 mg testosterone propionate in oil. In May 8 further males were treated in the same way. Between June and November 20 males whose hearts were being used in another study were tested with prolactin to see if there was any indication of responsiveness.
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Endo • 1975 Vol 97 • No 4
NASSAR ET AL.
1010
Prop 1
o
Prop 2
260
220 o
%of control
0
180 0 0 0
8
o
0 0
40 -
0 0 0
00 Amplitude
JL
Rate
at 15 seconds
FIG. 2. The results of 12 definitive experiments on adult male rabbit hearts in winter. At time zero 50 ng/ml prolactin was added to the perfusing fluid; at prop 1 50 /xg propanolol was injected, and at prop 2 20 fig propanolol was injected. The responses are shown as mean (±SEM) percentage changes from the amplitudes and rates immediately prior to the addition of ovine prolactin to the perfusates. The figure on the left shows the percentage changes in rate and amplitude 15 s after adding prolactin to the perfusate. The open circles represent what we have called type-A responses and are further detailed in the upper 2 figures on the right. The dark circles represent what we have called type-B responses and they are further described in the lower 2 figures on the right.
Results
fell into each group: in the 12 definitive experiments, for example, there were 7 typeAdult males in January and February and in A responses and 5 type-B ones. There were October and November. All 33 animals no differences in the starting heart rates tested in these months responded to pro- between the 2 groups: in hearts with type-A lactin with clear-cut changes in amplitude responses the mean starting rate was 144 and rate. There appeared to be 2 response beats/min ± 16.5 (SD) while in those with patterns (Fig. 2). One group (A) responded type-B responses it was 144 beats/ with very rapid rises in both rate and ampli- min ± 24.3. tude: an amplitude peak was reached within In the preliminary experiments it was the first 30 s with thereafter a fall to a pla- shown that on switching back to a control teau. The second group (B) responded with a Ringer-Locke solution the effect of promuch smaller and slower rise in rate and lactin disappeared within about 5 min. the initial amplitude peak was absent. All 5 reserpinized males showed type-B Approximately half the untreated animals responses. The starting heart rate in these
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PROLACTIN AND THE HEART animals was somewhat lower than in the others (124 ± 17.3). In both untreated and reserpinized animals the effects of prolactin were completely abolished by propanolol. The amplitude of contraction was restored to control levels but did not fall below these (Fig. 2) indicating that, in this preparation, the dose used had no direct myocardial depressant action. In preliminary experiments on hearts not treated with prolactin the propanolol had no effect on contraction amplitude. The heart rates after propanolol were slightly lower than in the control period which is consistent with the idea that even in the isolated heart there may be some endogenous catecholamine drive. Adult males tested from May to September. None of the 20 male hearts tested during this period showed any response to prolactin. The same batch of prolactin was used throughout and no changes were made in the composition of the perfusing fluid. In this period the same batch of prolactin continued to have its usual effect on arterioles (9). The loss of responsiveness between May and September and its restoration in October and November cannot therefore be accounted for by changes in procedure or in the potency of the prolactin used. Adult females. One untreated adult female out of 12 responded with a B-type pattern but apart from in this single animal, prolactin failed to have any cardiac chronotropic or inotropic effects. None of the 4 testosterone-treated females showed any change in rate but two showed a slight increase of contraction amplitude of less than 15% in response to prolactin. Prepubertal males. None of the 4 untreated prepubertal males showed any response to prolactin. None of the 4 testosteronetreated animals showed any change in rate but 2 showed small increases in amplitude of less than 15%.
1011 Discussion
The results demonstate that prolactin in a concentration of 50 ng/ml can have rapid inotropic and chronotropic actions on adult male rabbit hearts in winter but that this effect disappears in summer. All untreated prepubertal males and 11 out of 12 untreated females failed to show any response to prolactin even though they were tested in winter. After testosterone pretreatment of 4 adult females and 4 prepubertal males, half the animals in each group showed small inotropic responses to prolactin without any change in rate. Sexual differences in cardiac behavior are well established. In humans there are clear differences in male and female susceptibility to cardiac disease (13). In rats there are similar sexual differences in susceptibility to isoproterenol-induced myocardial infarction (14). The bases for these differences are unknown. One possible explanation of the current findings may be that adult male rabbit hearts, but not female or prepubertal male ones, have prolactin receptors. If so, the binding characteristics and mode of action of these receptors must be very different from those studied in other tissues such as the mammary gland and liver. They appear capable of producing a response within seconds and on removal of prolactin from the perfusing fluid the effect is terminated within minutes. Receptors with these characteristics might not be identified by the methods currently being used for study of prolactin receptors (15-17). The factors which determine the emergence of prolactin responsiveness in the heart would appear to be complex. In males in winter the difference between prepubertal animals and adults suggests the possibility of a dependence on testicular or perhaps adrenal hormones. The short course of testosterone given to prepubertal animals led to the emergence of a small inotropic effect of prolactin in 2 out of 4.
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1012
Enclo < 1975 Vol 97 . No 4
NASSAR ETAL.
It is possible that longer courses of treatment may produce responses more typical of those seen in adult males and this is currently under investigation. The fact that 1 out of 12 of the untreated adult females responded to prolactin and the effect of testosterone treatment also indicate that females are not absolutely barred from developing responsiveness. Again the possibility that gonadal or adrenal steroids (or perhaps even synthetic steroids given to human females) may be able to induce prolactin responsiveness deserves fuller investigation. The current experiments allow us to do little more than speculate about the seasonal changes in the males. The rabbits were obtained from commercial breeders prior to use. Their diets showed seasonal variations, they were exposed to natural changes in the photoperiod and they were housed in conditions which attenuated rather than abolished seasonal changes in temperature. Changes in any one of these factors might be important and again further experimentation is required. The work of Meier and his colleagues in sub-mammalian vertebrates has convincingly demonstrated dramatic seasonal variations in metabolic responses to prolactin and has indicated that changes in the photoperiod are a major factor (3,18,19). Others working with domestic animals have demonstrated marked seasonal variations in prolactin secretion with much higher values being reached in summer (20,21). In view of this background our own results are not particularly surprising. The responses seen in the male hearts in winter appeared to fall into 2 groups. One (type A) was characterized by very rapid rises in rate and amplitude with an initial peak and the other (type B) with much slower changes without the initial amplitude peak (Fig. 2). It seems possible that the 2 groups are not rigidly separate but if this is so it in no way affects the main conclusion that prolactin can have marked
inotropic and chronotropic actions on male hearts in winter. The animals in the 2 groups did not differ from one another in any other obvious way. Experiments were performed at various times of day between 10.00 and 18.00 h, but there was no correlation between the time of death and the response type. The actions of prolactin might be due to the release of catecholamines from endogenous cardiac stores, to a direct action of prolactin on the heart or to a mixture of the two. In the 5 animals treated with reserpine, which is thought to act by depleting catecholamine stores, only type-B responses were seen. This raises the possibility that the type-B responses might be direct effects of prolactin while the type-A ones might be due to a mixture of direct prolactin and indirect catecholamine effects. This cannot, at this stage, be more than a tentative suggestion; reserpine treatment as well as depleting catecholamine stores elevates prolactin levels (22,23) and this in itself could have influenced the result. In all the experiments in which it was used propanolol abolished both the inotropic and chronotropic actions of prolactin. Propanolol is a drug which blocks beta receptors but in addition it has membranestabilizing actions (24). The design of this study does not enable a decision to be made as to which action of propanol was responsible for the effect. Acknowledgments We acknowledge the financial support provided by the Leverhulme Trust, the Ernest Hart Fund of the British Medical Association, Hoechst and CibaGeigy. The ovine prolactin was kindly provided by the National Institute of Arthritis and MetabolicDiseases, Bethesda, Maryland, USA.
References 1. Weinstock, M., and S. Shoham, Nature 251: 427, 1974. 2. Dawson, W., B. A. Hems worth, and M. A. Stockham, J Pharm Pharmac 17: 183, 1965. 3. Meier, A. H., Gen Camp Endocrinol Supp 3: 499, 1972.
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PROLACTIN AND THE HEART 4. Nicoll, C. S., in Pasteels, J. L., and C. Robyn (eds), Human Prolactin, Excerpta Medica, Amsterdam, 1973, p. 119. 5. Hanwell, A., and J. L. Linzell, J Physiol 226: 24P, 1972a. 6. , and J Endocrinol 53: lvii, 1972b. 7. , and J Physiol 233: 93, 1973. 8. Guyton, A. C , C. E. Jones, and T. G. Coleman, Circulatory Physiology, ed 2, W. B. Saunders, Co., Philadelphia, 1973. 9. Manku, M. S., B. A. Nassar, and D. F. Horrobin, Lancet 2: 991, 1973. 10. Nassar, B. A., M. S. Manku, J. D. Reed, M. Tynan, D. F. Horrobin, Brit Med J 2: 27, 1974. 11. Langendorff, O., Pfliiger's Arch ges Physiol 61: 291, 1895. 12. Andrew, B. L., Experimental Physiology, ed. 9, Churchill Livingston, London, 1972. 13. Schornagel, H., and F. R. Ruttner, Pathol Microbiol 30: 517, 1967. 14. Wexler, B. C , and G. W. Kittinger, Circul Res 13: 159, 1963.
1013
15. Shiu, R. P., P. A. Kelly, and H. G. Friesen, Science 180: 968, 1973. 16. Posner, B. I., P. A. Kelly, R. P. Shiu, and H. G. Friesen, Endocrinology 95: 521, 1974. 17. Kelly, P. A., B. I. Posner, J. Tsushima, and H. G. Friesen, Endocrinology 95: 532, 1974. 18. Meier, A. H., D. D. Martin, and R. MacGregor, Science 173: 1240, 1971. 19. , T. N. Trobec, M. M. Joseph, and T. M. John, Proc Soc Exp Biol Med 137: 408, 1971. 20. Koprowski, J. A., and H. A. Tucker, Endocrinology 92: 1480, 1973. 21. Schams, D., and V. Reinhardt, Horm Res 5: 217, 1974. 22. Ratner, A., P. K. Talwalker, and J. Meites, Endocrinology 77: 315, 1965. 23. Meites, J., and J. A. Clemens, Vitam Horm 30: 165, 1972. 24. Nickerson, M., In Goodman, L. S., and A. Gilman (eds.), The Pharmacological Basis of Therapeutics, ed. 4, Macmillan, New York, 1970, p. 556.
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ABSTRACT. Rabbit hearts perfused by the Langendorff technique were studied. The addition of ovine prolactin (NIH-P-S-10) to the perfusate in a concentration of 50 ng/ml produced rapid increases in both the amplitude and rate of contraction in 33 adult male hearts studied in winter. Prepubertal male animals showed no response, and only 1 out of 12 adult females responded. Pretreatment for 10 days with 2.5 mg/day testosterone propionate led to minimal inotropic but not chronotropic responses in 2 out of 4 prepubertal males and 2 out of 4 adult females to prolactin. Clear responses to
S
EXUAL, seasonal, and temporal variations in responsiveness to hormones have recently attracted attention (1-4). We report here sexual and seasonal variations in the responses of the heart to prolactin. These experimental findings and the principles they illustrate are likely to be of considerable significance in the design and interpretation of studies of both animals and man. Prolactin seems to be in large part responsible for the increase in cardiac output associated with lactation (5-7). This does not prove that prolactin can have a direct cardiac effect since changes in the peripheral circulation will also produce changes in cardiac output (8). In isolated preparations of male rat superior mesenteric vascular beds, ovine prolactin in a concentration of 50 ng/ml potentiated arteriolar responses to noradrenaline and angiotensin without itself causing vasoconstriction (9). Concentrations of 200 ng/ml or over, in contrast, inhibited the effects of the vasoconstrictor agents. Depending on the selectivity of these actions both with respect to the vascular bed concerned and to the relative effects on resistance and capacitance vessels, it is Received March 17, 1975.
possible to conceive of mechanisms whereby either concentration of prolactin could increase venous return and enhance cardiac output. In isolated male rat hearts 50 ng/ml ovine prolactin increased the heart rate while 200 ng/ml slowed it down: both concentrations produced dysrhythmias (10). This paper demonstrates that 50 ng/ml ovine prolactin has inotropic and chronotropic actions on adult male rabbit hearts in winter but not in summer and that female and prepubertal male hearts are unresponsive to the hormone throughout the year. Methods and Materials Rabbits were killed by a blow on the head and the hearts were rapidly removed and perfused with oxygenated Ringer-Locke solution using the Langendorff technique (11,12). (This involves retrograde cannulation of the ascending aorta: the retrograde movement of the perfusate closes the aortic valves, and the fluid passes into the coronary arteries: no circulation passes through the chambers of the hearts (Fig. 1).) Contractions were recorded by means of a hook through the tip of the heart connected to an isometric force transducer. The amplitude and frequency (using an instantaneous rate meter) of the contractions were monitored using a moving paper recorder. After being attached to the perfusion system, the heart was allowed to
1008
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1009
PROLACTIN AND THE HEART stabilise for a 20 min period: hearts which showed dysrhythmias or which failed to achieve a stable rate and amplitude (no more than ±10% variation in eitlier parameter during the last 10 min of the period) were discarded without further testing. After this initial control period the perfusate was then changed from plain Ringer-Locke to a similar solution containing 50 ng/ml ovine prolactin (NIH-P-S-10). If there was no response to the prolactin the experiment was discontinued after 10 min. If there was a response, 3 min after switching to the prolactin solution a single injection of 50/x.g propanol ("Inderal," IC1) was injected into the perfusing cannula. The manufacturer's maximum recommended iv dose in humans is 10 mg: if cardiac plasma output of the human is 2500 ml/min this represents 400 /xg per 100 ml/min. The flow through our rabbit hearts was usually in the region of 30-35 ml/min giving a propanolol dose between 120-140 fxg per 100 ml/min. After a further 3 min a further 20 /xg of propanol was injected. Since prolactin is prepared from tissue which may contain vasoactive peptides and amines such as epinephrine, norepinephrine, dopamine, vasopressin, and oxytocin it is important to consider the possibility that the hormone could have been contaminated with these substances. The superior mesenteric artery of the rat is exquisitely sensitive to the vasoconstrictor actions of epinephrine, norepinephrine, and vasopressin: concentrations of the prolactin up to 5 fxg/m\ failed to have any even transient pressor effect in this preparation, and we therefore consider it extremely unlikely that the cardiac effects of 50 ng/ml prolactin could have been explained by one of these 3 substances. We also tested the direct effects of 5 ng/ml dopamine and oxytocin in the hearts on the assumption that a greater than 10% by weight contamination of the prolactin by such small molecular weight substances would be highly improbable given the methods used for the purification of protein hormones of mol wt about 20,000. Oxytocin in this concentration had no effect. Dopamine produced very small (less than 10%) increases in heart rate and contraction amplitude. However the effects of dopamine like those of epinephrine and norepinephrine occurred equally on male and female hearts. In the past 5 years, this department, in the course of student classes, has tested catecholamine
Perfusion
Cannula
Ascending aorta
Closed aortic valve
Coronary artery
Left ventricle
FIG. 1. Schematic diagram of LangendorfFs technique of cardiac perfusion. The perfusion fluid enters the aorta retrogradely and then, because of the closure of the aortic valves, passes along the coronary arteries. No fluid is pumped through the chambers in the usual way.
effects on about 200 male and female rabbits; no sex differences have been noted. The following Dutch rabbits were used in January and February: a) Eight adult males (at least 3 months post-pubertal) in preliminary experiments during which an appropriate protocol was established, b) 12 adult males in definitive experiments conducted as described, c) 5 adult males treated twice daily for 3 days with 0.5 mg reserpine subcutaneously, d) 4 untreated prepubertal males, e) 4 prepubertal males treated daily for 10 days with 2.5 mg testosterone propionate given subcutaneously in oil, f) 12 untreated adult females, and g) 4 adult females treated daily for 10 days with 2.5 mg testosterone propionate in oil. In May 8 further males were treated in the same way. Between June and November 20 males whose hearts were being used in another study were tested with prolactin to see if there was any indication of responsiveness.
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Endo • 1975 Vol 97 • No 4
NASSAR ET AL.
1010
Prop 1
o
Prop 2
260
220 o
%of control
0
180 0 0 0
8
o
0 0
40 -
0 0 0
00 Amplitude
JL
Rate
at 15 seconds
FIG. 2. The results of 12 definitive experiments on adult male rabbit hearts in winter. At time zero 50 ng/ml prolactin was added to the perfusing fluid; at prop 1 50 /xg propanolol was injected, and at prop 2 20 fig propanolol was injected. The responses are shown as mean (±SEM) percentage changes from the amplitudes and rates immediately prior to the addition of ovine prolactin to the perfusates. The figure on the left shows the percentage changes in rate and amplitude 15 s after adding prolactin to the perfusate. The open circles represent what we have called type-A responses and are further detailed in the upper 2 figures on the right. The dark circles represent what we have called type-B responses and they are further described in the lower 2 figures on the right.
Results
fell into each group: in the 12 definitive experiments, for example, there were 7 typeAdult males in January and February and in A responses and 5 type-B ones. There were October and November. All 33 animals no differences in the starting heart rates tested in these months responded to pro- between the 2 groups: in hearts with type-A lactin with clear-cut changes in amplitude responses the mean starting rate was 144 and rate. There appeared to be 2 response beats/min ± 16.5 (SD) while in those with patterns (Fig. 2). One group (A) responded type-B responses it was 144 beats/ with very rapid rises in both rate and ampli- min ± 24.3. tude: an amplitude peak was reached within In the preliminary experiments it was the first 30 s with thereafter a fall to a pla- shown that on switching back to a control teau. The second group (B) responded with a Ringer-Locke solution the effect of promuch smaller and slower rise in rate and lactin disappeared within about 5 min. the initial amplitude peak was absent. All 5 reserpinized males showed type-B Approximately half the untreated animals responses. The starting heart rate in these
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PROLACTIN AND THE HEART animals was somewhat lower than in the others (124 ± 17.3). In both untreated and reserpinized animals the effects of prolactin were completely abolished by propanolol. The amplitude of contraction was restored to control levels but did not fall below these (Fig. 2) indicating that, in this preparation, the dose used had no direct myocardial depressant action. In preliminary experiments on hearts not treated with prolactin the propanolol had no effect on contraction amplitude. The heart rates after propanolol were slightly lower than in the control period which is consistent with the idea that even in the isolated heart there may be some endogenous catecholamine drive. Adult males tested from May to September. None of the 20 male hearts tested during this period showed any response to prolactin. The same batch of prolactin was used throughout and no changes were made in the composition of the perfusing fluid. In this period the same batch of prolactin continued to have its usual effect on arterioles (9). The loss of responsiveness between May and September and its restoration in October and November cannot therefore be accounted for by changes in procedure or in the potency of the prolactin used. Adult females. One untreated adult female out of 12 responded with a B-type pattern but apart from in this single animal, prolactin failed to have any cardiac chronotropic or inotropic effects. None of the 4 testosterone-treated females showed any change in rate but two showed a slight increase of contraction amplitude of less than 15% in response to prolactin. Prepubertal males. None of the 4 untreated prepubertal males showed any response to prolactin. None of the 4 testosteronetreated animals showed any change in rate but 2 showed small increases in amplitude of less than 15%.
1011 Discussion
The results demonstate that prolactin in a concentration of 50 ng/ml can have rapid inotropic and chronotropic actions on adult male rabbit hearts in winter but that this effect disappears in summer. All untreated prepubertal males and 11 out of 12 untreated females failed to show any response to prolactin even though they were tested in winter. After testosterone pretreatment of 4 adult females and 4 prepubertal males, half the animals in each group showed small inotropic responses to prolactin without any change in rate. Sexual differences in cardiac behavior are well established. In humans there are clear differences in male and female susceptibility to cardiac disease (13). In rats there are similar sexual differences in susceptibility to isoproterenol-induced myocardial infarction (14). The bases for these differences are unknown. One possible explanation of the current findings may be that adult male rabbit hearts, but not female or prepubertal male ones, have prolactin receptors. If so, the binding characteristics and mode of action of these receptors must be very different from those studied in other tissues such as the mammary gland and liver. They appear capable of producing a response within seconds and on removal of prolactin from the perfusing fluid the effect is terminated within minutes. Receptors with these characteristics might not be identified by the methods currently being used for study of prolactin receptors (15-17). The factors which determine the emergence of prolactin responsiveness in the heart would appear to be complex. In males in winter the difference between prepubertal animals and adults suggests the possibility of a dependence on testicular or perhaps adrenal hormones. The short course of testosterone given to prepubertal animals led to the emergence of a small inotropic effect of prolactin in 2 out of 4.
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1012
Enclo < 1975 Vol 97 . No 4
NASSAR ETAL.
It is possible that longer courses of treatment may produce responses more typical of those seen in adult males and this is currently under investigation. The fact that 1 out of 12 of the untreated adult females responded to prolactin and the effect of testosterone treatment also indicate that females are not absolutely barred from developing responsiveness. Again the possibility that gonadal or adrenal steroids (or perhaps even synthetic steroids given to human females) may be able to induce prolactin responsiveness deserves fuller investigation. The current experiments allow us to do little more than speculate about the seasonal changes in the males. The rabbits were obtained from commercial breeders prior to use. Their diets showed seasonal variations, they were exposed to natural changes in the photoperiod and they were housed in conditions which attenuated rather than abolished seasonal changes in temperature. Changes in any one of these factors might be important and again further experimentation is required. The work of Meier and his colleagues in sub-mammalian vertebrates has convincingly demonstrated dramatic seasonal variations in metabolic responses to prolactin and has indicated that changes in the photoperiod are a major factor (3,18,19). Others working with domestic animals have demonstrated marked seasonal variations in prolactin secretion with much higher values being reached in summer (20,21). In view of this background our own results are not particularly surprising. The responses seen in the male hearts in winter appeared to fall into 2 groups. One (type A) was characterized by very rapid rises in rate and amplitude with an initial peak and the other (type B) with much slower changes without the initial amplitude peak (Fig. 2). It seems possible that the 2 groups are not rigidly separate but if this is so it in no way affects the main conclusion that prolactin can have marked
inotropic and chronotropic actions on male hearts in winter. The animals in the 2 groups did not differ from one another in any other obvious way. Experiments were performed at various times of day between 10.00 and 18.00 h, but there was no correlation between the time of death and the response type. The actions of prolactin might be due to the release of catecholamines from endogenous cardiac stores, to a direct action of prolactin on the heart or to a mixture of the two. In the 5 animals treated with reserpine, which is thought to act by depleting catecholamine stores, only type-B responses were seen. This raises the possibility that the type-B responses might be direct effects of prolactin while the type-A ones might be due to a mixture of direct prolactin and indirect catecholamine effects. This cannot, at this stage, be more than a tentative suggestion; reserpine treatment as well as depleting catecholamine stores elevates prolactin levels (22,23) and this in itself could have influenced the result. In all the experiments in which it was used propanolol abolished both the inotropic and chronotropic actions of prolactin. Propanolol is a drug which blocks beta receptors but in addition it has membranestabilizing actions (24). The design of this study does not enable a decision to be made as to which action of propanol was responsible for the effect. Acknowledgments We acknowledge the financial support provided by the Leverhulme Trust, the Ernest Hart Fund of the British Medical Association, Hoechst and CibaGeigy. The ovine prolactin was kindly provided by the National Institute of Arthritis and MetabolicDiseases, Bethesda, Maryland, USA.
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