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Mechanism of the effect of urethane on the secretion of prolactin in the male rat
Life Sciences, Vol. 29, pp. 1515-1522 Printed in the U.S.A.
Pergamon Pres
MECHANISM OF THE EFFECT OF URETHANE ON THE SECRETION OF PROLACTIN IN THE MALE RAT P.R. Findell, B.R. Larsen, B. Benson and D.E. Blask Department of Anatomy, University of Arizona Tucson, Arizona 85724 (Received in final form August 4, 1981) Summary A subcutaneous injection of urethane (200 mg/lO0 g body wt.) into adult male rats resulted in a significant increase in serum prolactin (PRL) at 30 minutes. Subsequent measurements at 60, 90 and 120 minutes postinjection revealed a marked and rapid decrease in serum PRL to levels significantly lower than those of unanesthetized animals. The administration of the dopamine antagonist pimozide (8, 40 or 200 ~g) 30 minutes after urethane injection elevated serum PRL levels in a dose-dependent manner and thus prevented the urethaneinduced depression in serum PRL observed at 60 minutes postinjection. Hypothalamic synthesis of 14C-dopamine from its precursor 14C-tyrosine was measured in both urethane-anesthetized and unanesthetized rats. The synthesis of hypothalamic dopamine was dramatically increased in the urethane-anesthetized animals as compared to newly synthesized hypothalamic dopamine levels in the unanesthetized controls. These results indicate that the PRLinhibitory effects of urethane anesthesia in the rat may be exerted through increased dopaminergic activity. In a variety of species, including the rat, increased PRL secretion is induced in response to the stress of surgery and anesthetics. As a result, large variations in baseline values of radioimmunologically measurable serum and plasma PRL are frequently encountered and make the interpretation of experimental data difficult. Two hours following its administration, urethane results in a stabilization of plasma PRL levels (I). For this reason this anesthetic has been used extensively in studies dealing with the control of PRL secretion. Previous studies have suggested that urethane administration modifies the central control of PRL release. For example, urethane anesthesia has been shown to depress significantly plasma PRL to levels that could not be further suppressed by L-DOPA administration (2). Urethane is purported to have similar effects on rat growth hormone secretion (3). Burnet and Wakerly (4) found that although the suckling-induced rise in serum PRL in rats treated with urethane was not abolished, its rate was much slower compared with the dramatic PRL surge displayed by unanesthetized animals. An anesthetic dose of urethane also affects luteinizing hormone-releasing hormone release and blocks spontaneous ovulation in the rat when ad~inistered during or just prior to proestrus (5). The above evidence raises certain questions about the suitability of urethane anesthesia in neuroendocrine studies reouiring the measurement of PRL 0024-3205/81/151515-08502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
1516
Urethane and Prolactin Secretion
Vol. 29, No.
15, 1981
This possibility prompted us to investigate in a more comprehensive manner the effects of urethane on the secretion of PRL in the rat. Materials and Methods Animals Eight-week-old male Sprague-Dawley rats (250-300 g body weight) were obtained from Camm Laboratory Animals (Wayne, New Jersey) and housed two per cage under conditions of controlled lighting (12h light:12h dark) and temperature (23°C). Laboratory chow and water were provided to the animals ad libitum. Animals were anesthetized with urethane (Sigma Chemical Co.) administered via s.c. injection at a dosage of 200 mg/100 g body wt. Experimental Design Exp. i. Seven animals were anesthetized with urethane while seven control animals received single s.c. injections of physiological saline. One hour after injection each animal was sacrificed by decapitation. Truncal blood was collected in glass tubes and allowed to clot at room temperature. The clotted blood was then centrifuged and the serum was stored frozen until PRL radioimmunoassay. Exp. 2. Two groups of six animals each were treated as described above and sacrificed by decapitation two hours after injections. Truncal blood was collected and treated as described above. Exp. 3. Five animals were sacrificed by decapitation to provide unanesthetized control serum PRL values. Five additional animals were anesthetized with urethane and blood samples (0.7 ml) taken via jugular venipuncture at 30, 60, 90 and 120 minutes postinjection. Exp. 4. Forty-two animals were divided into six groups of seven animals each. One group of animals was irmnediately sacrificed by decapitation to provide unanesthetized control serum PRL values. Animals in the remaining five groups were anesthetized with urethane and blood samples (0.7 ml) were taken via jugular venipuncture at 60 minutes. In addition, animals of four of the five urethane-anesthetized groups received one of the following treatments administered via the external jugular vein at 30 minutes: 0.i ml of 0.05 M tartaric acid or 0.i ml of 0.05 ~! tartaric acid containing either 8, 40 or 200 ~g of the dopamine antagonist pimozide (McNeil Labs). The animals in the remaining urethane-anesthetized group received no additional treatment. Blood samples were treated as described above. Exp. 5. Sixteen animals were divided into two groups of eight animals each. Seventy-two hours prior to testing, one group of animals was fitted with intra-atrial cannulae (silastic medical grade tubing, 0.025 in. I.D. by 0.047 in. O.D.) via the external jugular vein. To maintain their patency, the cannulae were flushed with physiological saline daily. On the morning of the experiment, animals of the remaining group were anesthetized with urethane via s.c. injection while the cannulated animals received a s.c. injection of s a l i n e One hour after the injections, animals of both groups received 0.2 ml of saline containing 35 ~Ci of 14C-tyrosine (specific activity 473 m C i / m ~ l ) (Research Products International Corp.) administered via intra-atrial catheters in the unanesthetized animals and via injection into the surgically exposed external jugular vein in the anesthetized animals. One hour after the 14C-tyrosine administration the animals were sacrificed by decapitation, truncal blood was collected and the hypothalami removed for determination of 14C incorporation into dopamine (DA) and norepinephrine (NE).
Vol. 29, No.
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Urethane and Prolactin Secretion
1517
Determination of Hypothalamic 14C-DA and 14C-NE The hypothalami were dissected via cuts made caudal to the optic chiasm, rostral to the mammillary bodies, parasagittally through the hypothalamic fissures formed with the temporal lobes, and horizontally 3 mm dorsal to the ventral surface of the median eminence (avg. tissue wt. = 30 mg). Radiolabeled DA and NE were extracted from the tissue following the procedure of Ogasahara et al. (6), with minor modifications. In~nediately after removal, each hypothalamus was sonicated (Kontes microultrasonic cell disrupter, maximum setting) for 60 seconds in 3.0 ml of 3% perchloric acid containing 0.2% ethylenediamine tetraacetic acid and 0.2% ascorbic acid (0°C). The suspensions were centrifuged at 12,350 x g for 15 minutes at 4oc, and supernatent fractions removed and stored at -120oc until final processing. Upon thawing, 2.0 ~g of DA and NE (Sigma Chemical Co.) were added to each perchlorate solution and the pH raised to 6.5 ± 0.2 using 4.0 or 0 . 1 M potassium hydroxide. Each solution was placed on a 0.6 x 9 cm bed of Bio-Rex 70 (Bio-Rad Laboratories) equilibrated with 0.1 M sodium phosphate at pH 6.0. The column was first washed with 10 ml of water. NE and DA were then eluted from the column with 0 . 0 1 M sodium phosphate (pH 6.0) containing 1.5% boric acid and 0.10 M sodium phosphate (pH 6.0) containing 4.0% boric acid, respectively, The elution of the DA and NE standards was monitored by subjecting 50 ~i of each collected I ml fraction to high pressure liquid chromatography (HPLC). A Waters Associates Model 6000A chromatography pump, a U6K injector, and a 0.39 x 30 cm alkyl phenyl column coupled with a ~ d e l 440 absorbance detector were used for the HPLC. NE and DA were eluted with 0 . 0 1 M acetic acid (2 ml/min flow rate) and detected using the maximum sensitivity of the absorbance detector (ABS at 280 monitored). NE and DA were pooled according to the elution profile indicated by the HPLC chromatograms. Each pooled sample was lyophilized, reconstituted in 2 ml water and subjected to scintillation counting. PRL Radioimmunoassay Duplicate 50 ~i aliquots of each serum sample were assayed for immunoreactive PRL using a kit supplied by NIAMDD. Prolactin levels were expressed in terms of reference preparation RP-I. Statistics Student's t-tests and, where appropriate, analyses of variance were used to assess significant differences between groups. Results The results of experiments i and 2 are shown in Table I. PRL concentrations in truncal blood collected after decapitation were reduced 78 and 76% in mlimals anesthetized with urethane for 60 and 120 minutes, respectively, as compared to PRL concentrations in truncal blood collected from unanesthetized control rats. TABLE 1
Exp. 1 Exp. 2
N
Unanesthet. Controls PRL (ng/ml)
7 6
18.6 + 3.1 a 5.5 ± 1.3
N
Urethane 60 min. PRL (ng/ml)
7
4.0 ± i.I b
N
Urethane 120 min. PRL (ng/ml)
% Reduction from Control Levels
6
1.3 ± 0.3 c
78 76
N = the number of animals per group
b p < 0.005 versus control
a Mean ± S.E.M.
c p < 0.01
versus control
1518
Urethane
and P r o l a c t i n
Secretion
Vol.
29, No.
15,
1981
1201101009080. o)
70._1
nO. E
60-
2 a) (/)
50-
4030 20 10-
@-
FIG.
@-
1
Serum PRL levels in male rats at various times after the a d m i n i s t r a tion of u r e t h a n e (U). Each b a r represents the mean ± S.E.M. for five animals, a = p < 0.05 and b,c,d = p < 0.01 versus the value for u n a n e s t h e t i z e d controls.
In e x p e r i m e n t 3 PRL c o n c e n t r a t i o n s in blood samples collected via j u g u l a r v e n i p u n c t u r e from u r e t h a n e - t r e a t e d animals d e m o n s t r a t e d an 83% e l e v a t i o n 30 m i n u t e s after urethane a d m i n i s t r a t i o n compared to the PRL c o n c e n t r a t i o n in t r u n c a l b l o o d collected from u n a n e s t h e t i z e d control animals (Fig. i). Subsequent serum PRL m e a s u r e m e n t s in the a n e s t h e t i z e d animals d e m o n s t r a t e d a marked, rapid decrease in serum PRL at 60, 90 and 120 minutes following ure-thane injection. These values represent a 78 to 87% reduction in serum PRL in u r e t h a n e - a n e s t h e t i z e d animals compared w i t h s e r u m PRL values in u n a n e s t h e t i z e d control rats.
Vol.
29, No.
15,
1981
Urethane
and Prolactin
Secretion
1519
600 -
.~C
500-
E
400-
_1
nr 12..
E 2
300-
CO
200"
100"
FIG.
2
Serum PRL levels in male rats anesthetized with urethane (U) and treated with 8, 40 or 200 ~g pimozide (P). Pimozide injections were made 30 minutes after urethane administration. Blood samples were taken via jugular venipuncture 60 minutes after urethane administration. Each bar represents the mean ± S.E.M. for 7 animals, a = p < 0.025 and b,c = p < 0.001 versus the value for unanesthetized controls.
Similarly, in experiment 4 (Fig. 2) PRL concentrations in rats anesthetized w i t h urethane for 60 minutes were significantly lower than PRL concentrations in truncal blood collected from unanesthetized control animals. As shown in Fig. 2, the administration of 8 ~g of pimozide slightly elevated serum PRL levels in animals anesthetized with urethane for 60 minutes. Increasingamount~ of pimozide, viz. 40 or 200 ~g, resulted in a 265 or 538% increase in serum PRL levels respectively, compared to serum PRL levels in unanesthetized control animals. The animals which received vehicle administration of 0.05 M tartaric acid (not shown in Fig. 2) showed serum PRL levels at 60 minutes not
1520
Urethane
and Prolactin
Secretion
significantly different from serum PRL values receiving no further treatment.
Vol.
29, No.
of u r e t h a n e - a n e s t h e t i z e d
1.2-
15,
1981
animals
~6.0
1.11.0-
0.90.8-
///J ///J 1111 sl/s
1
I//J ///I ///I ///I ///J i i i /
0.7-.
111/ / i / /
E 0.6-
"5.0
-4.0 3
-o -n r-
-3.0
=_. n
3_
0.5-
0
0.4-J
-2,0
0.3'
0.1~-
0.2-
"1.O
FIG.
3
H y p o t h a l a m i c synthesis of 14C-DA and 14C-NE and serum PRL levels in rats anesthetized with urethane. Each bar represents the mean ± S.E.M. of eight animals, a = p < 0.001 versus unanesthetized controls; b = p < 0.005 versus mean PRL levels in unanesthetized controls.
In experiment 5 h y p o t h a l a m i c levels of newly synthesized 14C-DA were compared in u r e t h a n e - a n e s t h e t i z e d and unanesthetized rats (Fig. 3). The mean quantity of 14C-DA extracted from the hypothalami of animals anesthetized with urethane for 120 minutes was 76% greater than that extracted from the hypothalami of unanesthetized control animals. No significant difference was observed in the amount of newly synthesized 14C-NE e x t r a c t e d from h y p o t h a l a m i of u r e t h a n e - a n e s t h e t i z e d animals vs. unanesthetized control rats. PRL concentrations in truncal blood collected after decapitation were reduced 70% in the animals a n e s t h e t i z e d with urethane for 120 minutes as compared to PRL concentrations in truncal blood collected from the unanesthetized animals.
Vol. 29, No.
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U rethane and Prolactin Secretion
1521
Discussion The data presented in this and other studies indicate that urethane anesthesia modifies the control of PRL release in the rat. PRL concentrations in serial blood samples collected via jugular venipuncture demonstrated that following a transient increase in serum levels of PRL, urethane-anesthetized rats have significantly lower serum PRL levels compared with unanesthetized controls. This method of blood sampling, i.e., serial bleedings without fluid replacement, has been shown to cause a decrease in plasma PRL (7). However, in this study PRL concentrations in truncal blood collected after decapitation also demonstrated dramatically lower serum PRL levels in animals anesthetized with urethane. Since PRL concentrations in serial blood samples and truncal blood collected after decapitation were reduced 70% and 76% respectively, below those levels observed in unanesthetized controls, it does not appear that blood sampling without fluid replacement significantly decreased serum PRL in this investigation. The low levels of serum PRL observed in the urethaneanesthetized animals in the present study corroborate the results of a previous study by Smythe and Lazarus (2) who demonstrated that plasma PRL levels in male rats were significantly suppressed two hours after urethane administration. An abundance of evidence now exists which demonstrates that DA is the hypothalamic factor responsible for the physiological inhibition of PRL secretion (8). Axons of cells located in the arcuate and periventricular nuclei of the mediobasal hypothalamus project to the external lamina of the median eminence (tuberoinfundibular system) and terminate in the proximity of the capillary plexus of the hypophysial portal system. DA released from the terminals of these nerves is carried to the anterior pituitary gland via hypophysial portal blood vessels where it is thought to activate receptors located on PRL secreting cells. Activation of these receptors inhibits the secretion of PRL and results in decreased serum concentrations of PRL. In the present study, blockade of the DA receptors pimozide resulted in increased serum PRL concentrations anesthetized animals. This DA antagonist prevented the depression of serum PRL levels and indicates that these activated in the urethane-anesthetized animal.
with the DA antagonist in urethaneurethane-induced receptors are tonically
The administration of amphetamine has been demonstrated to inhibit PRL secretion in rats (9) and appears to exert its effects through the release of newly synthesized DA. Increased DA levels in hypophysial portal blood have been observed after amphetamine administration (I0). Similarly, our results indicate that urethane inhibited PRL secretion by increasing the hypothalamic synthesis of DA. This was evidenced by the presence of greater amounts of newly synthesized 14C-DA in the hypothalami of urethane-anesthetized animals compared to that present in hypothalamic tissue of unanesthetized animals. Pilotte and co-workers (14) reported that male rats under urethane anesthesia had lower serum PRL levels and greater concentrations of DA in pituitary stalk plasma than animals anesthetized with pentobarbital. In possible disagreement with evidence for increased DA synthesis under urethane anesthesia provided by our data, these authors reported that DA turnover in urethane-anesthetized animals was not significantly different from that in pentobarbital-anesthetized animals. However, since Pilotte et al. did not measure DA turnover in unanesthetized control animals a comparison of their results to results presented in this manuscript is difficult. Serotonin-containing neurons located in the raphe nuclei of the brainstem, and norepinephrine-containing neurons located in the locus ceruleus, project to the tuberoinfundibular system and are believed also to regulate PRL secretion
1522
Urethane and Prolactin Secretion
Vol. 29, No. 15, 1981
(14). NE and serotonin do not appear to be required for the tonic inhibition of PRL release, but may regulate DA neuronal activity and thus mediate phasic changes in PRL secretion. The possibility exists that urethane may exert its PRL inhibiting effects through changes in the activity of this extrinsic innervation to DA neurons. In the present study, urethane did not appear to affect NE synthesis in the hypothalamus. Hypothalamic levels of newly synthesized 14C-NE in urethane-anesthetized animals were not significantly different from levels in unanesthetized rats, The inhibitory influence of urethane on PRL secretion in male rats may perhaps be eliminated by steroidal pretreatment. In support of such an hypothesis a study by Boyd and co-workers (I) provided evidence that plasma PRL levels in male rats pretreated with estradiol and progesterone were not significantly depressed 60 and 120 minutes after urethane administration. Estrogens have been demonstrated to be potent stimulators of PRL secretion in the male rat (12). Moreover, estrogen has been shown to have an antidopaminergic effect. In male rats, the inhibition of the morphine-induced PRL secretion by subcutaneous administration of DA was significantly blocked in animals pretreated with estradiol benzoate (13). These findings may explain why the inhibitory effect of urethane on PRL secretion is eliminated by steroidal pretreatment, since the mechanism of urethane's inhibitory influence on PRL secretion appears to be through increased dopaminergic activity. Lawson and Gala (7,11) have reported that urethane had no significant depressive effect on plasma PRL in ovariectomized or ovariectomized-estrogen treated female rats. Therefore, there may exist a sexual dimorphism in the response to urethane when animals are treated with estrogen. In conclusion, it is probable that when administered to experimental animals urethane increases hypothalamic dopaminergic activity and diminished PRL release results from increased levels of DA delivered to the anterior pituitary via hypophysial portal blood vessels. Acknowledsements This work was supported by N.I.H. Grant #HD-08759. The technical assistance of Carla A. LaPorte is gratefully acknowledged. References i. 2. 3. 4. 5. 6. 7. 8. 9. 10, ii, 12. 13. 14. 15.
A.E. BOYD, IiI, E. SPENCER, I.M.D. JACKSON and S. REICHLIN, Endocrinology 99 861 (1976). G.A. SMYTHE and L. LAZARUS, Endocrinology 93 147 (1973). R. COLLU, F. FRASCHINI, P. VISCONTI and L. MARTINI, Endocrinology 9 0 1231 (1972). F.R. BURNET and J.B. WAKERLEY, Endocrinology 70 429 (1976). C.A. BLAKE and C.H. SAWYER, Endocrinology 91 87 (1972). S. OGASAHARA, T. MANDAI, A. YAMATODANI, T. WATANAB, H. WADA and T. SEKI, J. Chromatogr. 180 119 (1979). D.M. LAWSON and R.R. GALA, J. Endocrinol. 62 75 (1974). J.A. CLEMENS, C.J. SHAAR and E.B. SMALSTIG, Fed. Proc. 39(11) 2907 (1980). J.A. CLEMENS and R.W. FULLER, Life Sci. 24 2077 (1979). G.A. GUDELSKY and J.C. PORTER, Endocrinology 104 583 (1979). D.M. LAWSON and R.R. GALA, J. Endocrinol. 66 151 (1975). A. DE LEAN and F. LABRIE, Am. J. Physiol. 223(3) 235 (1977). L. FERLAND, F. LABRIE, C. EUVRARD and J.P. RAYNAUD, Mol. Cell. Endocrinol. 14 199 (1979). N.S. PILOTTE, G.A. GUDELSKY and J.C. PORTER, Brain Res. 193 284 (1980). R.I. WEINER and W.F. GANONG, Physiol. Rev. 58 905 (1978).
Pergamon Pres
MECHANISM OF THE EFFECT OF URETHANE ON THE SECRETION OF PROLACTIN IN THE MALE RAT P.R. Findell, B.R. Larsen, B. Benson and D.E. Blask Department of Anatomy, University of Arizona Tucson, Arizona 85724 (Received in final form August 4, 1981) Summary A subcutaneous injection of urethane (200 mg/lO0 g body wt.) into adult male rats resulted in a significant increase in serum prolactin (PRL) at 30 minutes. Subsequent measurements at 60, 90 and 120 minutes postinjection revealed a marked and rapid decrease in serum PRL to levels significantly lower than those of unanesthetized animals. The administration of the dopamine antagonist pimozide (8, 40 or 200 ~g) 30 minutes after urethane injection elevated serum PRL levels in a dose-dependent manner and thus prevented the urethaneinduced depression in serum PRL observed at 60 minutes postinjection. Hypothalamic synthesis of 14C-dopamine from its precursor 14C-tyrosine was measured in both urethane-anesthetized and unanesthetized rats. The synthesis of hypothalamic dopamine was dramatically increased in the urethane-anesthetized animals as compared to newly synthesized hypothalamic dopamine levels in the unanesthetized controls. These results indicate that the PRLinhibitory effects of urethane anesthesia in the rat may be exerted through increased dopaminergic activity. In a variety of species, including the rat, increased PRL secretion is induced in response to the stress of surgery and anesthetics. As a result, large variations in baseline values of radioimmunologically measurable serum and plasma PRL are frequently encountered and make the interpretation of experimental data difficult. Two hours following its administration, urethane results in a stabilization of plasma PRL levels (I). For this reason this anesthetic has been used extensively in studies dealing with the control of PRL secretion. Previous studies have suggested that urethane administration modifies the central control of PRL release. For example, urethane anesthesia has been shown to depress significantly plasma PRL to levels that could not be further suppressed by L-DOPA administration (2). Urethane is purported to have similar effects on rat growth hormone secretion (3). Burnet and Wakerly (4) found that although the suckling-induced rise in serum PRL in rats treated with urethane was not abolished, its rate was much slower compared with the dramatic PRL surge displayed by unanesthetized animals. An anesthetic dose of urethane also affects luteinizing hormone-releasing hormone release and blocks spontaneous ovulation in the rat when ad~inistered during or just prior to proestrus (5). The above evidence raises certain questions about the suitability of urethane anesthesia in neuroendocrine studies reouiring the measurement of PRL 0024-3205/81/151515-08502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
1516
Urethane and Prolactin Secretion
Vol. 29, No.
15, 1981
This possibility prompted us to investigate in a more comprehensive manner the effects of urethane on the secretion of PRL in the rat. Materials and Methods Animals Eight-week-old male Sprague-Dawley rats (250-300 g body weight) were obtained from Camm Laboratory Animals (Wayne, New Jersey) and housed two per cage under conditions of controlled lighting (12h light:12h dark) and temperature (23°C). Laboratory chow and water were provided to the animals ad libitum. Animals were anesthetized with urethane (Sigma Chemical Co.) administered via s.c. injection at a dosage of 200 mg/100 g body wt. Experimental Design Exp. i. Seven animals were anesthetized with urethane while seven control animals received single s.c. injections of physiological saline. One hour after injection each animal was sacrificed by decapitation. Truncal blood was collected in glass tubes and allowed to clot at room temperature. The clotted blood was then centrifuged and the serum was stored frozen until PRL radioimmunoassay. Exp. 2. Two groups of six animals each were treated as described above and sacrificed by decapitation two hours after injections. Truncal blood was collected and treated as described above. Exp. 3. Five animals were sacrificed by decapitation to provide unanesthetized control serum PRL values. Five additional animals were anesthetized with urethane and blood samples (0.7 ml) taken via jugular venipuncture at 30, 60, 90 and 120 minutes postinjection. Exp. 4. Forty-two animals were divided into six groups of seven animals each. One group of animals was irmnediately sacrificed by decapitation to provide unanesthetized control serum PRL values. Animals in the remaining five groups were anesthetized with urethane and blood samples (0.7 ml) were taken via jugular venipuncture at 60 minutes. In addition, animals of four of the five urethane-anesthetized groups received one of the following treatments administered via the external jugular vein at 30 minutes: 0.i ml of 0.05 M tartaric acid or 0.i ml of 0.05 ~! tartaric acid containing either 8, 40 or 200 ~g of the dopamine antagonist pimozide (McNeil Labs). The animals in the remaining urethane-anesthetized group received no additional treatment. Blood samples were treated as described above. Exp. 5. Sixteen animals were divided into two groups of eight animals each. Seventy-two hours prior to testing, one group of animals was fitted with intra-atrial cannulae (silastic medical grade tubing, 0.025 in. I.D. by 0.047 in. O.D.) via the external jugular vein. To maintain their patency, the cannulae were flushed with physiological saline daily. On the morning of the experiment, animals of the remaining group were anesthetized with urethane via s.c. injection while the cannulated animals received a s.c. injection of s a l i n e One hour after the injections, animals of both groups received 0.2 ml of saline containing 35 ~Ci of 14C-tyrosine (specific activity 473 m C i / m ~ l ) (Research Products International Corp.) administered via intra-atrial catheters in the unanesthetized animals and via injection into the surgically exposed external jugular vein in the anesthetized animals. One hour after the 14C-tyrosine administration the animals were sacrificed by decapitation, truncal blood was collected and the hypothalami removed for determination of 14C incorporation into dopamine (DA) and norepinephrine (NE).
Vol. 29, No.
15, 1981
Urethane and Prolactin Secretion
1517
Determination of Hypothalamic 14C-DA and 14C-NE The hypothalami were dissected via cuts made caudal to the optic chiasm, rostral to the mammillary bodies, parasagittally through the hypothalamic fissures formed with the temporal lobes, and horizontally 3 mm dorsal to the ventral surface of the median eminence (avg. tissue wt. = 30 mg). Radiolabeled DA and NE were extracted from the tissue following the procedure of Ogasahara et al. (6), with minor modifications. In~nediately after removal, each hypothalamus was sonicated (Kontes microultrasonic cell disrupter, maximum setting) for 60 seconds in 3.0 ml of 3% perchloric acid containing 0.2% ethylenediamine tetraacetic acid and 0.2% ascorbic acid (0°C). The suspensions were centrifuged at 12,350 x g for 15 minutes at 4oc, and supernatent fractions removed and stored at -120oc until final processing. Upon thawing, 2.0 ~g of DA and NE (Sigma Chemical Co.) were added to each perchlorate solution and the pH raised to 6.5 ± 0.2 using 4.0 or 0 . 1 M potassium hydroxide. Each solution was placed on a 0.6 x 9 cm bed of Bio-Rex 70 (Bio-Rad Laboratories) equilibrated with 0.1 M sodium phosphate at pH 6.0. The column was first washed with 10 ml of water. NE and DA were then eluted from the column with 0 . 0 1 M sodium phosphate (pH 6.0) containing 1.5% boric acid and 0.10 M sodium phosphate (pH 6.0) containing 4.0% boric acid, respectively, The elution of the DA and NE standards was monitored by subjecting 50 ~i of each collected I ml fraction to high pressure liquid chromatography (HPLC). A Waters Associates Model 6000A chromatography pump, a U6K injector, and a 0.39 x 30 cm alkyl phenyl column coupled with a ~ d e l 440 absorbance detector were used for the HPLC. NE and DA were eluted with 0 . 0 1 M acetic acid (2 ml/min flow rate) and detected using the maximum sensitivity of the absorbance detector (ABS at 280 monitored). NE and DA were pooled according to the elution profile indicated by the HPLC chromatograms. Each pooled sample was lyophilized, reconstituted in 2 ml water and subjected to scintillation counting. PRL Radioimmunoassay Duplicate 50 ~i aliquots of each serum sample were assayed for immunoreactive PRL using a kit supplied by NIAMDD. Prolactin levels were expressed in terms of reference preparation RP-I. Statistics Student's t-tests and, where appropriate, analyses of variance were used to assess significant differences between groups. Results The results of experiments i and 2 are shown in Table I. PRL concentrations in truncal blood collected after decapitation were reduced 78 and 76% in mlimals anesthetized with urethane for 60 and 120 minutes, respectively, as compared to PRL concentrations in truncal blood collected from unanesthetized control rats. TABLE 1
Exp. 1 Exp. 2
N
Unanesthet. Controls PRL (ng/ml)
7 6
18.6 + 3.1 a 5.5 ± 1.3
N
Urethane 60 min. PRL (ng/ml)
7
4.0 ± i.I b
N
Urethane 120 min. PRL (ng/ml)
% Reduction from Control Levels
6
1.3 ± 0.3 c
78 76
N = the number of animals per group
b p < 0.005 versus control
a Mean ± S.E.M.
c p < 0.01
versus control
1518
Urethane
and P r o l a c t i n
Secretion
Vol.
29, No.
15,
1981
1201101009080. o)
70._1
nO. E
60-
2 a) (/)
50-
4030 20 10-
@-
FIG.
@-
1
Serum PRL levels in male rats at various times after the a d m i n i s t r a tion of u r e t h a n e (U). Each b a r represents the mean ± S.E.M. for five animals, a = p < 0.05 and b,c,d = p < 0.01 versus the value for u n a n e s t h e t i z e d controls.
In e x p e r i m e n t 3 PRL c o n c e n t r a t i o n s in blood samples collected via j u g u l a r v e n i p u n c t u r e from u r e t h a n e - t r e a t e d animals d e m o n s t r a t e d an 83% e l e v a t i o n 30 m i n u t e s after urethane a d m i n i s t r a t i o n compared to the PRL c o n c e n t r a t i o n in t r u n c a l b l o o d collected from u n a n e s t h e t i z e d control animals (Fig. i). Subsequent serum PRL m e a s u r e m e n t s in the a n e s t h e t i z e d animals d e m o n s t r a t e d a marked, rapid decrease in serum PRL at 60, 90 and 120 minutes following ure-thane injection. These values represent a 78 to 87% reduction in serum PRL in u r e t h a n e - a n e s t h e t i z e d animals compared w i t h s e r u m PRL values in u n a n e s t h e t i z e d control rats.
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15,
1981
Urethane
and Prolactin
Secretion
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600 -
.~C
500-
E
400-
_1
nr 12..
E 2
300-
CO
200"
100"
FIG.
2
Serum PRL levels in male rats anesthetized with urethane (U) and treated with 8, 40 or 200 ~g pimozide (P). Pimozide injections were made 30 minutes after urethane administration. Blood samples were taken via jugular venipuncture 60 minutes after urethane administration. Each bar represents the mean ± S.E.M. for 7 animals, a = p < 0.025 and b,c = p < 0.001 versus the value for unanesthetized controls.
Similarly, in experiment 4 (Fig. 2) PRL concentrations in rats anesthetized w i t h urethane for 60 minutes were significantly lower than PRL concentrations in truncal blood collected from unanesthetized control animals. As shown in Fig. 2, the administration of 8 ~g of pimozide slightly elevated serum PRL levels in animals anesthetized with urethane for 60 minutes. Increasingamount~ of pimozide, viz. 40 or 200 ~g, resulted in a 265 or 538% increase in serum PRL levels respectively, compared to serum PRL levels in unanesthetized control animals. The animals which received vehicle administration of 0.05 M tartaric acid (not shown in Fig. 2) showed serum PRL levels at 60 minutes not
1520
Urethane
and Prolactin
Secretion
significantly different from serum PRL values receiving no further treatment.
Vol.
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of u r e t h a n e - a n e s t h e t i z e d
1.2-
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1981
animals
~6.0
1.11.0-
0.90.8-
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1
I//J ///I ///I ///I ///J i i i /
0.7-.
111/ / i / /
E 0.6-
"5.0
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-3.0
=_. n
3_
0.5-
0
0.4-J
-2,0
0.3'
0.1~-
0.2-
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FIG.
3
H y p o t h a l a m i c synthesis of 14C-DA and 14C-NE and serum PRL levels in rats anesthetized with urethane. Each bar represents the mean ± S.E.M. of eight animals, a = p < 0.001 versus unanesthetized controls; b = p < 0.005 versus mean PRL levels in unanesthetized controls.
In experiment 5 h y p o t h a l a m i c levels of newly synthesized 14C-DA were compared in u r e t h a n e - a n e s t h e t i z e d and unanesthetized rats (Fig. 3). The mean quantity of 14C-DA extracted from the hypothalami of animals anesthetized with urethane for 120 minutes was 76% greater than that extracted from the hypothalami of unanesthetized control animals. No significant difference was observed in the amount of newly synthesized 14C-NE e x t r a c t e d from h y p o t h a l a m i of u r e t h a n e - a n e s t h e t i z e d animals vs. unanesthetized control rats. PRL concentrations in truncal blood collected after decapitation were reduced 70% in the animals a n e s t h e t i z e d with urethane for 120 minutes as compared to PRL concentrations in truncal blood collected from the unanesthetized animals.
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U rethane and Prolactin Secretion
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Discussion The data presented in this and other studies indicate that urethane anesthesia modifies the control of PRL release in the rat. PRL concentrations in serial blood samples collected via jugular venipuncture demonstrated that following a transient increase in serum levels of PRL, urethane-anesthetized rats have significantly lower serum PRL levels compared with unanesthetized controls. This method of blood sampling, i.e., serial bleedings without fluid replacement, has been shown to cause a decrease in plasma PRL (7). However, in this study PRL concentrations in truncal blood collected after decapitation also demonstrated dramatically lower serum PRL levels in animals anesthetized with urethane. Since PRL concentrations in serial blood samples and truncal blood collected after decapitation were reduced 70% and 76% respectively, below those levels observed in unanesthetized controls, it does not appear that blood sampling without fluid replacement significantly decreased serum PRL in this investigation. The low levels of serum PRL observed in the urethaneanesthetized animals in the present study corroborate the results of a previous study by Smythe and Lazarus (2) who demonstrated that plasma PRL levels in male rats were significantly suppressed two hours after urethane administration. An abundance of evidence now exists which demonstrates that DA is the hypothalamic factor responsible for the physiological inhibition of PRL secretion (8). Axons of cells located in the arcuate and periventricular nuclei of the mediobasal hypothalamus project to the external lamina of the median eminence (tuberoinfundibular system) and terminate in the proximity of the capillary plexus of the hypophysial portal system. DA released from the terminals of these nerves is carried to the anterior pituitary gland via hypophysial portal blood vessels where it is thought to activate receptors located on PRL secreting cells. Activation of these receptors inhibits the secretion of PRL and results in decreased serum concentrations of PRL. In the present study, blockade of the DA receptors pimozide resulted in increased serum PRL concentrations anesthetized animals. This DA antagonist prevented the depression of serum PRL levels and indicates that these activated in the urethane-anesthetized animal.
with the DA antagonist in urethaneurethane-induced receptors are tonically
The administration of amphetamine has been demonstrated to inhibit PRL secretion in rats (9) and appears to exert its effects through the release of newly synthesized DA. Increased DA levels in hypophysial portal blood have been observed after amphetamine administration (I0). Similarly, our results indicate that urethane inhibited PRL secretion by increasing the hypothalamic synthesis of DA. This was evidenced by the presence of greater amounts of newly synthesized 14C-DA in the hypothalami of urethane-anesthetized animals compared to that present in hypothalamic tissue of unanesthetized animals. Pilotte and co-workers (14) reported that male rats under urethane anesthesia had lower serum PRL levels and greater concentrations of DA in pituitary stalk plasma than animals anesthetized with pentobarbital. In possible disagreement with evidence for increased DA synthesis under urethane anesthesia provided by our data, these authors reported that DA turnover in urethane-anesthetized animals was not significantly different from that in pentobarbital-anesthetized animals. However, since Pilotte et al. did not measure DA turnover in unanesthetized control animals a comparison of their results to results presented in this manuscript is difficult. Serotonin-containing neurons located in the raphe nuclei of the brainstem, and norepinephrine-containing neurons located in the locus ceruleus, project to the tuberoinfundibular system and are believed also to regulate PRL secretion
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(14). NE and serotonin do not appear to be required for the tonic inhibition of PRL release, but may regulate DA neuronal activity and thus mediate phasic changes in PRL secretion. The possibility exists that urethane may exert its PRL inhibiting effects through changes in the activity of this extrinsic innervation to DA neurons. In the present study, urethane did not appear to affect NE synthesis in the hypothalamus. Hypothalamic levels of newly synthesized 14C-NE in urethane-anesthetized animals were not significantly different from levels in unanesthetized rats, The inhibitory influence of urethane on PRL secretion in male rats may perhaps be eliminated by steroidal pretreatment. In support of such an hypothesis a study by Boyd and co-workers (I) provided evidence that plasma PRL levels in male rats pretreated with estradiol and progesterone were not significantly depressed 60 and 120 minutes after urethane administration. Estrogens have been demonstrated to be potent stimulators of PRL secretion in the male rat (12). Moreover, estrogen has been shown to have an antidopaminergic effect. In male rats, the inhibition of the morphine-induced PRL secretion by subcutaneous administration of DA was significantly blocked in animals pretreated with estradiol benzoate (13). These findings may explain why the inhibitory effect of urethane on PRL secretion is eliminated by steroidal pretreatment, since the mechanism of urethane's inhibitory influence on PRL secretion appears to be through increased dopaminergic activity. Lawson and Gala (7,11) have reported that urethane had no significant depressive effect on plasma PRL in ovariectomized or ovariectomized-estrogen treated female rats. Therefore, there may exist a sexual dimorphism in the response to urethane when animals are treated with estrogen. In conclusion, it is probable that when administered to experimental animals urethane increases hypothalamic dopaminergic activity and diminished PRL release results from increased levels of DA delivered to the anterior pituitary via hypophysial portal blood vessels. Acknowledsements This work was supported by N.I.H. Grant #HD-08759. The technical assistance of Carla A. LaPorte is gratefully acknowledged. References i. 2. 3. 4. 5. 6. 7. 8. 9. 10, ii, 12. 13. 14. 15.
A.E. BOYD, IiI, E. SPENCER, I.M.D. JACKSON and S. REICHLIN, Endocrinology 99 861 (1976). G.A. SMYTHE and L. LAZARUS, Endocrinology 93 147 (1973). R. COLLU, F. FRASCHINI, P. VISCONTI and L. MARTINI, Endocrinology 9 0 1231 (1972). F.R. BURNET and J.B. WAKERLEY, Endocrinology 70 429 (1976). C.A. BLAKE and C.H. SAWYER, Endocrinology 91 87 (1972). S. OGASAHARA, T. MANDAI, A. YAMATODANI, T. WATANAB, H. WADA and T. SEKI, J. Chromatogr. 180 119 (1979). D.M. LAWSON and R.R. GALA, J. Endocrinol. 62 75 (1974). J.A. CLEMENS, C.J. SHAAR and E.B. SMALSTIG, Fed. Proc. 39(11) 2907 (1980). J.A. CLEMENS and R.W. FULLER, Life Sci. 24 2077 (1979). G.A. GUDELSKY and J.C. PORTER, Endocrinology 104 583 (1979). D.M. LAWSON and R.R. GALA, J. Endocrinol. 66 151 (1975). A. DE LEAN and F. LABRIE, Am. J. Physiol. 223(3) 235 (1977). L. FERLAND, F. LABRIE, C. EUVRARD and J.P. RAYNAUD, Mol. Cell. Endocrinol. 14 199 (1979). N.S. PILOTTE, G.A. GUDELSKY and J.C. PORTER, Brain Res. 193 284 (1980). R.I. WEINER and W.F. GANONG, Physiol. Rev. 58 905 (1978).