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Prolactin stimulates dopamine release from the rat corpus striatum in the absence of extra-cellular calcium
Neurascience Letters, 134 (1991) 1-4
1
© 1991 ElsevierScientificPublishers Ireland Ltd. All rights reserved0304-3940/91/$03.50 NSL 08255
Prolactin stimulates dopamine release from the rat corpus striatum in the absence of extra-cellular calcium N.J. L a p i n g 1, D . E . D l u z e n 2 a n d V.D. R a m i r e z a 1Gerontology Center, U.S.C., Los Angeles, CA 90089-0191 (U.S.A.), 2Department of Anatomy, North Eastern Ohio University, College of Medicine, Rootstown, 0H44272-9989 (U.S.A.) and 3Department of Physiology and Biophysics, Urbana, 1L 61801 (U.S.A.)
(Received6 August 1991;Revisedversion received10 September 1991;Accepted 13 September1991) Key words: Amphetamine;Calcium; Corpus striatum; Dopamine; Prolactin;Rat
Prolactin (PRL) increased basal dopamine (DA) release and attenuated amphetamine (AMPH)-stimulated DA release in vitro from rat corpus striatum in a concentration-dependentmanner with 10-5 M PRL being the most effective.The effectsof PRL on DA release were enhanced in the absence of extracellularcalcium. PRL at 10-5 M did not alter the DA post-superfusion content of the striatal tissue. These results indicate that the stimulatory effectof PRL on basal DA release does not require extra-cellularcalcium and the inhibitory effecton AMPH-stimulated DA releaseis not due to depletionof DA stores.
Prolactin (PRL), a large protein hormone of pituitary origin, has a wide range o f effects on a number of behaviors including maternal [17], feeding [19], sexual [7], and learning behavior [1]. Activation of specific behaviors is dependent on the dose of PRL used since it has been shown that lesser doses of PRL can induce yawning behavior in rats [13, 14] while at high, hyperprolactinemic doses, P R L induces grooming behavior [8]. It has been assumed that P R L affects behaviors by acting on the CNS and there is good evidence in support of such an hypothesis. For example, an interaction between central dopamine receptors and P R L was shown by demonstrating that P R L could antagonize bromocryptine-induced behaviors in cats [10]. In addition, specific P R L binding sites exist on the epithelial cells of the choroid plexus which could possibly play a role in transporting plasma P R L into the CSF [21], particularly since increases in plasma P R L are reflected by increases in CSF P R L [2, 15, 16]. PRL immunoreactive fibers have also been identified in the hypothalamus and to some extent in the corpus striatum [11, 23]. Finally, specific PRL binding sites in the hypothalamus, substantia nigra, and corpus striatum (CS) support a CNS site of action for PRL [6]. While most work involved in examining the effects of P R L upon the CNS have focused on the hypothalamus Correspondence: V.D. Ramirez, Department of Physiologyand Biophysics, 524 Burrill Hall, 407 S. Goodwin, Urbana, IL 61801, U.S.A.
and tuberoinfundibular dopamine system, it is clear that this hormone is quite capable of altering dopaminergic function in the CS of the nigro-striatal dopamine system. Within the CS P R L has been shown to increase dopamine (DA) turnover [22], potentiate electrically stimulated DA release [20], and increase endogenous DA release in vitro and in vivo [3-5]. Moreover, PRL has been shown to decrease acetylcholine turnover in the striatum and septum, areas where DA is known to inhibit acetylcholine release, presumably via stimulation of DA release [24]. While it is clear that P R L is effective in releasing DA, the mechanism by which P R L releases DA, including such basic questions as calcium requirements, have not been addressed. Therefore, the purpose of this report is to examine some of the mechanisms which govern P R L regulation o f basal as well as amphetamine (AMPH)-stimulated DA release from the CS. Adult (3-4 months) male albino Holtzman (SpragueDawley) rats were used in the present experiments. The animals were group-housed in clear plastic cages and maintained under a 14/10 h light/dark cycle (lights on at 05.00 h). Purina lab chow and water were furnished ad libitum. On the day of an experiment one rat was decapitated (09.00-10.00 h) and CS from both hemispheres were removed and placed in medium on ice. The CS were anatomically defined by a coronal cut through the optic chiasm, and dorsal and lateral cuts within the perimeter
of the corpus callosum to a depth of approximately 2 mm. The CS were further dissected into approximately 0.5 × 0.5 × 0.5 m m tissue fragments and approximately 3-7 mg of CS tissue fragments were then placed in each chamber. The superfusion medium was described before [12]. The modified superfusion chamber constructed from the barrel of a one ml plastic syringe has been described previously [12]. The mini-chambers were maintained at 37°C and effluent samples were collected at 10 minute intervals at a flow rate near 10/d/min. Note that the combined CS tissue removed from one animal was sufficient to fill 8 chambers. In this way, the CS from one animal was able to be subjected to several or all treatments, thereby generating an n of one for each treatment at the same time. A total of 8 rats were used in these experiments. The CS tissue fragments were superfused with either normal K r e b s - R i n g e r - p h o s p h a t e (KRP) medium (control: 120 m M NaCl, 4.8 m M KCI, 2.6 m M CaC12, 1.2 m M MgSO4, 10.2 m M Na2HPO4, 1.8 m M NaH2PO4, 0.18% glucose at pH = 7.4), or K R P medium containing ovine P R L at concentrations of l0 -7, l 0 - 6 , o r 10 - 5 M (lyophilized, Sigma). Also, CS tissue from male rats was superfused with either in calcium-free medium (plus l m M E G T A as a chealating agent) or calcium-free medium with P R L at 10 -5 M. In initial experiments 1 m M E D T A was used instead of E G T A to chelate calcium. However, E D T A appeared to be toxic to the tissue for it blocked AMPH-induced D A release, a calcium independent process, and raised basal D A release. E G T A on the other hand did not block AMPH-induced D A release. For all experiments, after a 25 minute equilibration period using normal K R P , media were exchanged with treatment media and equilibrated for an additional 15 min. Then 12 samples were collected in tubes on ice at l0 minute intervals and assayed immediately for D A (see below). During sample 5, the control and treatment media were exchanged with identical media containing 10 p M A M P H . Following the superfusion, the tissue was weighed for each chamber. Each condition was replicated 4-8 times. The samples were analyzed for D A using high pressure liquid chromatography with electrochemical detection (HPLC-EC) and the data expressed as pg D A per mg of tissue per minute as described before [12]. To determine statistical significance among the different concentrations of PRL, samples 1-5 (basal DA release) and samples 6-12 (stimulated D A release) were compared between treatments by two-way A N O V A ' s (sample × P R L concentration). The effect of E G T A on PRLinduced D A release was also analyzed by two-way A N O V A (sample × PRL, in the presence of EGTA).
Effect on
in
vitro
Basal
~
40
E
30
of PRL DA
release
AMPH stimulated
+
D.
•
20
W
@
"~
lO
< ~
o Control
10 -7 M Dose
10 -6 M
10-5M
PRL
Fig. 1. S u m m a r y of basal and AMPH-stimulated in vitro D A release rates from corpus striatum perfused with control medium or medium containing prolactin at 10 7 M, 10 -6 M, or 10 -s M. Open bars indicate basal D A release; mean release rate of samples 1 5 _ S.E.M. I0/~M A M P H was infused during sample 5. Hatched bars indicate A M P H stimulated D A release; mean release rate of samples 6-12 + S.E.M. N u m b e r s in bars indicate number of replications. *P < 0.05 vs control; + P < 0 . 0 5 vs 10 -7 M PRL.
A summary of basal and AMPH-stimulated D A release from CS tissue fragments in the presence of increasing concentrations of ovine P R L is shown in Fig. 1. In the presence of PRL, basal D A release was enhanced in a concentration-dependent manner. Basal D A release (mean + S.E.M. of samples 1-5) under the control condition was 6.0_+0.8 pg x mg -1 × min -1, while increasing concentrations of P R L ( 1 0 - 7 , 10 - 6 , l 0 - 5 M ) elevated basal D A release to 6.6___0.7, 11.1_2.3, and 12.8 _+2.1 * pg x m g - 1 x m i n - 1, respectively (*P < 0.05 vs control). In contrast to the effect of P R L on basal DA release, high concentrations of P R L attenuated A M P H stimulated DA release. Ten I~M A M P H released 29.5 _ 4.6 pg x m g - J × m i n - l D A under control conditions but increasing concentrations of P R L 0 0 -7, 10 -6, l0 -5 M) slightly raised and then attenuated D A release to 34.0_+4.8, 25.4_+4.0, and 18.8_2.9" p g x m g -1 × m i n -1 respectively (*P<0.05 v s 10 - 7 M PRL). DA content of post-superfused striatal tissue was 2.44_ 0.45 ng D A / m g tissue (wet weight) for controls and 2.07_+ 0.42 ng/mg for l0 -5 M P R L treated tissue. These content values failed to differ significantly. The effect of l0 -5 M P R L on D A release in the absence of extra-cellular calcium is shown in Table I. In the absence of extra-cellular calcium basal D A release was 7.6_+1.6 p g x m g - l x m i n - l , similar to controls (6.0_+0.8 p g × m g - I ×min-1). P R L was able to stimulate basal D A release in the absence of calcium (28.4_+2.5 p g x m g -1 × m i n - l ; P<0.0001). Note that
TABLE I PRL INDUCED DA RELEASE IN THE ABSENCE OF EXTRACELLULARCALCIUM Basal DA release AMPH-stimulated (pg × mg-t x min- i) DA release (pg x mg- 1x min- l) Control (2.6mM Ca2+) 10-5 M PRL (2.6 mM Ca2+) I mM EGTA 1 mM EGTA + 10-5 M PRL
6.0 __+0.8 (n=8)
29.5 ___4.6(n=8)
12.8 __+2.1 (n=8)* 18.8 __+2.9 (n=8)* 7.6 ___1.6 (n=4) 46.8 __+9.9 (n=4) 28.4 __+2.5 (n=4)*** 24.1 _+ 3.8 (n=4)*
0.05 vs control. **P<0.01 vs 1 mM EGTA. ***P<0.0001 vs 1 mM EGTA. *P <
this increase was twice as high as the stimulation evoked in the presence of calcium (Table I). P R L at 10 -5 M was also able to attenuate the AMPH-stimulated D A release in the absence of extra-cellular calcium (46.8 +9.9 vs 24.1___3.8 p g x m g - l x m i n -1, Ca2+-free control vs PRL/Ca2+-free; P < 0.003). Continuous administration of ovine P R L for 65 min at 10 -5 M concentration stimulated basal D A release from the CS of male rats. This supports previous findings that a 20-30 min pulse of ovine P R L can stimulate D A release from this structure as determined in vitro and in vivo [3-5, 20] and favors dopaminergic mediation of certain PRL-induced behaviors such as grooming and yawning [8, 13]. The higher concentration of P R L required for a significant effect compared to previous studies within this laboratory is probably due to differences in the administration of P R L (continuous vs. pulse), and in the assay for D A determination (radioenzymatic vs HPLC-EC). The present results indicate that P R L can exert a prolonged stimulatory action of basal dopamine release and also demonstrate that P R L attenuates AMPH-stimulated D A release from the CS of male rats under continuous exposure to this protein. The exact means by which P R L modulates D A release from CS tissue is not known. However, the present results provide some new information regarding potential mechanisms. First, P R L does not appear to directly stimulate D A synthesis in vitro as shown by post superfusion content and by total amount of D A released (basal + AMPH-stimulated) between control and 10 -5 M PRL. This supports previous findings that P R L stimulates D A synthesis in the median eminence but not in the striatum in vivo [18]. Second, our results clearly show that the P R L effects on basal and AMPH-stimulated D A release are independent of extracellular calcium and
apparently enhanced by the absence of extracellular calcium. These findings combined with previous data which showed that P R L release D A in the presence of tetrodotoxin [4], strongly suggest that P R L is exerting its effect directly upon dopaminergic terminals o f the CS without involving inflow of extracellular calcium ions or altering D A synthesis. It is possible that continuous exposure to P R L and the prolonged increase in basal D A release can deplete a hypothetical AMPH-sensitive D A storage pool sufficiently to result in an attenuated AMPH-stimulated D A response. However, the present results showed that postsuperfusion D A content levels were not affected by 1 0 - S M PRL. An alternate explanation is that P R L might interfere with the A M P H mediated D A release mechanism. Specifically, P R L might competitively interact with A M P H at D A reuptake sites. It is worth emphasizing that AMPH-induced D A release is a calcium-independent process as well [9] and thus further supports the above hypothesis. In summary, these data showed that P R L can release D A from the CS which is an extracellular calcium-independent process. Prolactin-induced attenuation of AMPH-induced D A release is probably not due to depletion of D A stores. The elusive nature of P R L binding sites in the rat striatum and the effects on D A release and behavior might be elucidated by examining interactions of P R L with the D A reuptake mechanism. This work was supported by N I H Grants PHS 5 732GM7143 to N.J.L., PHS 2 R01 HD14625 to V.D.R., and the Parkinson Disease Foundation and United Way of Stark County to D.E.D.
1 Banerjee, U., Influenceof pseudopregnancy and sex hormones on conditioned behavior in rats, Neuroendocrinology, 7 (1971) 278290. 2 Belchez,P.E., Ridley, R.M. and Baker, H.F., Studies on the accessibility of prolactin and growth hormone to brain: effect of opiate agonists on hormonelevelsin serial, simultaneous plasma and cerebral spinal fluid samples in rhesus monkey, Brain Res., 239 (1982) 310-314. 3 Chen, J.C., Ramirez, A.D. and Ramirez, V.D., Prolactin effect on In Vivo striatal dopaminergicactivity as estimated with push-pull perfusion in male rate. In R. Macleod, U., Scapagnini and M. Thorner (Eds.), Prolactin, Basic and Clinical Correlates, Springer, New York, 1985, pp. 523-532. 4 Chen, J.C. and Ramirez, V.D., Comparison of the effect of prolactin on the dopamine release from the rat dorsal and ventral striatum and from the mediobasal hypothalamus superfused in vitro, Eur. J. Pharmacol., 149 (1988) 1-8. 5 Chen, Y.F. and Ramirez, V.D., Prolactin stimulates the release of dopamine from male but not female striatal tissue superfused in vitro, Endocrinology, I 11 (1982) 1740-1742. 6 Di Carlo, R., Muccioli, G., Lando, D. and Belussi, G., Further evidence for the presence of specific binding sites for prolactin in the
7
8
9
10
11
12
13
14
rabbit brain. Preferential distribution in the hypothalamus and substantia nigra, Life Sci., 36 (1985) 375-382. Doherty, P.C., Baum, M.J. and Todd, R.B., Effects of chronic hyperprolactinemia on sexual arousal and erectile function in male rats, Neuroendocrinology, 42 (1986) 368-375. Drago, F., Canonico, P.L., Bitteti, R. and Scapagnini, U., Systemic and intraventricular prolaetin induces excessive grooming, Eur. J. Pharmacol., 65 (1980)457-458. Fischer, J.F. and Cho, A.K., Chemical release of dopamine from striatal homogenates: evidence for an exchange diffusion model, J. Pharmacol. Exp. Ther., 208 (1979) 203-209. Gonzalez-Lima, F., Velez, D. and Blanco, R., Antagonism of behavioral effects of bromoeriptine by prolactin in female cats, Behav. Neural. Biol., 49 (1988) 74-82. Harlan, R.E., Shivers, B.D., Fox, S.R., Kaplove, K.E., Schachter, B.S. and Pfaff, D.W., Distribution and partial characterization of immunoreactive prolactin in the rat brain, Neuroendocrinology, 49 (1989) 7-22. Laping, N.J., Dluzen, D.E. and Ramirez, V.D., Aging alters opiate modulation of potassium evoked DA release from the corpus striaturn of male rats. Neurobiol. Aging, 11 (1990) 395-399. Laping, N.J. and Ramirez, V.D., Prolactin induces yawning and the stretch-yawning-syndrome in young adult male rats, Horm. Behav., 20 (1986) 49-59. Laping, N.J. and Ramirez, V.D., Prolactin-induced yawning behavior requires and intact nigro-striatal dopamine system, Pharm. Biochem. Behav., 29 (1988) 59-62.
15 Login, I.S. and MacLeod, R.M., Prolactin in human rat serum and cerebrospinal fluid, Brain Res., 132 (1977) 477-483. 16 Martenez, N.D. and Herbert, J., Relationship between prolactin in the serum and cerebrospinal fluid of ovariectomized female rhesus monkeys, Neuroscience, 7 (1982) 2801-2812. 17 Moltz, H., Lubin, M., Leon, M. and Numan, M., Hormonal induction of maternal behavior in the ovariectomized nulliparous rat, Physiol. Behav., 5 (1970) 1373-1377. 18 Moore, K.E., Interactions between prolactin and dopaminergic neurons, Biol. Reprod., 36 (1987) 47-58. 19 Nicoll, C.S., Physiological actions of prolactin. In Handbook of Physiology, Vol. 7, Endocrinology, American Physiological Society, Washington, DC, 1974, pp. 253-292. 20 Perkins, N.A. and Westfall, T.C., The effect of prolactin on dopamine release from rat striatum and medial basal hypothalamus, Neuroscience, 3 (1978) 59-63. 21 Silverman, W.F., Walsh, R.J. and Posner, B.I., The ontogeny of prolactin binding sites in the rat choroid plexus, Dev. Brain Res., 24 (1986) 11-19. 22 Van Loon, G.R., Shum, A., George, S.R. and Shin, S.H., Prolactin increases the activity of tuberoinfundibular and nigrostriatal neurons, Brain Res. Bull., 10 (1982) 539-545. 23 Walsh, R.J., Posner, B.I., Kopriva, B.M. and Brawer, J.R., Prolactin binding sites in the rat brain, Science, 201 (1978) 1041-1043. 24 Wood, P.L., Cheney, D.L. and Costa, E., A prolactin action on acetyl choline metabolism in striatum, hippocampus, and thalamus, J. Neurochem., 34 (1980) 1053-1057.
1
© 1991 ElsevierScientificPublishers Ireland Ltd. All rights reserved0304-3940/91/$03.50 NSL 08255
Prolactin stimulates dopamine release from the rat corpus striatum in the absence of extra-cellular calcium N.J. L a p i n g 1, D . E . D l u z e n 2 a n d V.D. R a m i r e z a 1Gerontology Center, U.S.C., Los Angeles, CA 90089-0191 (U.S.A.), 2Department of Anatomy, North Eastern Ohio University, College of Medicine, Rootstown, 0H44272-9989 (U.S.A.) and 3Department of Physiology and Biophysics, Urbana, 1L 61801 (U.S.A.)
(Received6 August 1991;Revisedversion received10 September 1991;Accepted 13 September1991) Key words: Amphetamine;Calcium; Corpus striatum; Dopamine; Prolactin;Rat
Prolactin (PRL) increased basal dopamine (DA) release and attenuated amphetamine (AMPH)-stimulated DA release in vitro from rat corpus striatum in a concentration-dependentmanner with 10-5 M PRL being the most effective.The effectsof PRL on DA release were enhanced in the absence of extracellularcalcium. PRL at 10-5 M did not alter the DA post-superfusion content of the striatal tissue. These results indicate that the stimulatory effectof PRL on basal DA release does not require extra-cellularcalcium and the inhibitory effecton AMPH-stimulated DA releaseis not due to depletionof DA stores.
Prolactin (PRL), a large protein hormone of pituitary origin, has a wide range o f effects on a number of behaviors including maternal [17], feeding [19], sexual [7], and learning behavior [1]. Activation of specific behaviors is dependent on the dose of PRL used since it has been shown that lesser doses of PRL can induce yawning behavior in rats [13, 14] while at high, hyperprolactinemic doses, P R L induces grooming behavior [8]. It has been assumed that P R L affects behaviors by acting on the CNS and there is good evidence in support of such an hypothesis. For example, an interaction between central dopamine receptors and P R L was shown by demonstrating that P R L could antagonize bromocryptine-induced behaviors in cats [10]. In addition, specific P R L binding sites exist on the epithelial cells of the choroid plexus which could possibly play a role in transporting plasma P R L into the CSF [21], particularly since increases in plasma P R L are reflected by increases in CSF P R L [2, 15, 16]. PRL immunoreactive fibers have also been identified in the hypothalamus and to some extent in the corpus striatum [11, 23]. Finally, specific PRL binding sites in the hypothalamus, substantia nigra, and corpus striatum (CS) support a CNS site of action for PRL [6]. While most work involved in examining the effects of P R L upon the CNS have focused on the hypothalamus Correspondence: V.D. Ramirez, Department of Physiologyand Biophysics, 524 Burrill Hall, 407 S. Goodwin, Urbana, IL 61801, U.S.A.
and tuberoinfundibular dopamine system, it is clear that this hormone is quite capable of altering dopaminergic function in the CS of the nigro-striatal dopamine system. Within the CS P R L has been shown to increase dopamine (DA) turnover [22], potentiate electrically stimulated DA release [20], and increase endogenous DA release in vitro and in vivo [3-5]. Moreover, PRL has been shown to decrease acetylcholine turnover in the striatum and septum, areas where DA is known to inhibit acetylcholine release, presumably via stimulation of DA release [24]. While it is clear that P R L is effective in releasing DA, the mechanism by which P R L releases DA, including such basic questions as calcium requirements, have not been addressed. Therefore, the purpose of this report is to examine some of the mechanisms which govern P R L regulation o f basal as well as amphetamine (AMPH)-stimulated DA release from the CS. Adult (3-4 months) male albino Holtzman (SpragueDawley) rats were used in the present experiments. The animals were group-housed in clear plastic cages and maintained under a 14/10 h light/dark cycle (lights on at 05.00 h). Purina lab chow and water were furnished ad libitum. On the day of an experiment one rat was decapitated (09.00-10.00 h) and CS from both hemispheres were removed and placed in medium on ice. The CS were anatomically defined by a coronal cut through the optic chiasm, and dorsal and lateral cuts within the perimeter
of the corpus callosum to a depth of approximately 2 mm. The CS were further dissected into approximately 0.5 × 0.5 × 0.5 m m tissue fragments and approximately 3-7 mg of CS tissue fragments were then placed in each chamber. The superfusion medium was described before [12]. The modified superfusion chamber constructed from the barrel of a one ml plastic syringe has been described previously [12]. The mini-chambers were maintained at 37°C and effluent samples were collected at 10 minute intervals at a flow rate near 10/d/min. Note that the combined CS tissue removed from one animal was sufficient to fill 8 chambers. In this way, the CS from one animal was able to be subjected to several or all treatments, thereby generating an n of one for each treatment at the same time. A total of 8 rats were used in these experiments. The CS tissue fragments were superfused with either normal K r e b s - R i n g e r - p h o s p h a t e (KRP) medium (control: 120 m M NaCl, 4.8 m M KCI, 2.6 m M CaC12, 1.2 m M MgSO4, 10.2 m M Na2HPO4, 1.8 m M NaH2PO4, 0.18% glucose at pH = 7.4), or K R P medium containing ovine P R L at concentrations of l0 -7, l 0 - 6 , o r 10 - 5 M (lyophilized, Sigma). Also, CS tissue from male rats was superfused with either in calcium-free medium (plus l m M E G T A as a chealating agent) or calcium-free medium with P R L at 10 -5 M. In initial experiments 1 m M E D T A was used instead of E G T A to chelate calcium. However, E D T A appeared to be toxic to the tissue for it blocked AMPH-induced D A release, a calcium independent process, and raised basal D A release. E G T A on the other hand did not block AMPH-induced D A release. For all experiments, after a 25 minute equilibration period using normal K R P , media were exchanged with treatment media and equilibrated for an additional 15 min. Then 12 samples were collected in tubes on ice at l0 minute intervals and assayed immediately for D A (see below). During sample 5, the control and treatment media were exchanged with identical media containing 10 p M A M P H . Following the superfusion, the tissue was weighed for each chamber. Each condition was replicated 4-8 times. The samples were analyzed for D A using high pressure liquid chromatography with electrochemical detection (HPLC-EC) and the data expressed as pg D A per mg of tissue per minute as described before [12]. To determine statistical significance among the different concentrations of PRL, samples 1-5 (basal DA release) and samples 6-12 (stimulated D A release) were compared between treatments by two-way A N O V A ' s (sample × P R L concentration). The effect of E G T A on PRLinduced D A release was also analyzed by two-way A N O V A (sample × PRL, in the presence of EGTA).
Effect on
in
vitro
Basal
~
40
E
30
of PRL DA
release
AMPH stimulated
+
D.
•
20
W
@
"~
lO
< ~
o Control
10 -7 M Dose
10 -6 M
10-5M
PRL
Fig. 1. S u m m a r y of basal and AMPH-stimulated in vitro D A release rates from corpus striatum perfused with control medium or medium containing prolactin at 10 7 M, 10 -6 M, or 10 -s M. Open bars indicate basal D A release; mean release rate of samples 1 5 _ S.E.M. I0/~M A M P H was infused during sample 5. Hatched bars indicate A M P H stimulated D A release; mean release rate of samples 6-12 + S.E.M. N u m b e r s in bars indicate number of replications. *P < 0.05 vs control; + P < 0 . 0 5 vs 10 -7 M PRL.
A summary of basal and AMPH-stimulated D A release from CS tissue fragments in the presence of increasing concentrations of ovine P R L is shown in Fig. 1. In the presence of PRL, basal D A release was enhanced in a concentration-dependent manner. Basal D A release (mean + S.E.M. of samples 1-5) under the control condition was 6.0_+0.8 pg x mg -1 × min -1, while increasing concentrations of P R L ( 1 0 - 7 , 10 - 6 , l 0 - 5 M ) elevated basal D A release to 6.6___0.7, 11.1_2.3, and 12.8 _+2.1 * pg x m g - 1 x m i n - 1, respectively (*P < 0.05 vs control). In contrast to the effect of P R L on basal DA release, high concentrations of P R L attenuated A M P H stimulated DA release. Ten I~M A M P H released 29.5 _ 4.6 pg x m g - J × m i n - l D A under control conditions but increasing concentrations of P R L 0 0 -7, 10 -6, l0 -5 M) slightly raised and then attenuated D A release to 34.0_+4.8, 25.4_+4.0, and 18.8_2.9" p g x m g -1 × m i n -1 respectively (*P<0.05 v s 10 - 7 M PRL). DA content of post-superfused striatal tissue was 2.44_ 0.45 ng D A / m g tissue (wet weight) for controls and 2.07_+ 0.42 ng/mg for l0 -5 M P R L treated tissue. These content values failed to differ significantly. The effect of l0 -5 M P R L on D A release in the absence of extra-cellular calcium is shown in Table I. In the absence of extra-cellular calcium basal D A release was 7.6_+1.6 p g x m g - l x m i n - l , similar to controls (6.0_+0.8 p g × m g - I ×min-1). P R L was able to stimulate basal D A release in the absence of calcium (28.4_+2.5 p g x m g -1 × m i n - l ; P<0.0001). Note that
TABLE I PRL INDUCED DA RELEASE IN THE ABSENCE OF EXTRACELLULARCALCIUM Basal DA release AMPH-stimulated (pg × mg-t x min- i) DA release (pg x mg- 1x min- l) Control (2.6mM Ca2+) 10-5 M PRL (2.6 mM Ca2+) I mM EGTA 1 mM EGTA + 10-5 M PRL
6.0 __+0.8 (n=8)
29.5 ___4.6(n=8)
12.8 __+2.1 (n=8)* 18.8 __+2.9 (n=8)* 7.6 ___1.6 (n=4) 46.8 __+9.9 (n=4) 28.4 __+2.5 (n=4)*** 24.1 _+ 3.8 (n=4)*
0.05 vs control. **P<0.01 vs 1 mM EGTA. ***P<0.0001 vs 1 mM EGTA. *P <
this increase was twice as high as the stimulation evoked in the presence of calcium (Table I). P R L at 10 -5 M was also able to attenuate the AMPH-stimulated D A release in the absence of extra-cellular calcium (46.8 +9.9 vs 24.1___3.8 p g x m g - l x m i n -1, Ca2+-free control vs PRL/Ca2+-free; P < 0.003). Continuous administration of ovine P R L for 65 min at 10 -5 M concentration stimulated basal D A release from the CS of male rats. This supports previous findings that a 20-30 min pulse of ovine P R L can stimulate D A release from this structure as determined in vitro and in vivo [3-5, 20] and favors dopaminergic mediation of certain PRL-induced behaviors such as grooming and yawning [8, 13]. The higher concentration of P R L required for a significant effect compared to previous studies within this laboratory is probably due to differences in the administration of P R L (continuous vs. pulse), and in the assay for D A determination (radioenzymatic vs HPLC-EC). The present results indicate that P R L can exert a prolonged stimulatory action of basal dopamine release and also demonstrate that P R L attenuates AMPH-stimulated D A release from the CS of male rats under continuous exposure to this protein. The exact means by which P R L modulates D A release from CS tissue is not known. However, the present results provide some new information regarding potential mechanisms. First, P R L does not appear to directly stimulate D A synthesis in vitro as shown by post superfusion content and by total amount of D A released (basal + AMPH-stimulated) between control and 10 -5 M PRL. This supports previous findings that P R L stimulates D A synthesis in the median eminence but not in the striatum in vivo [18]. Second, our results clearly show that the P R L effects on basal and AMPH-stimulated D A release are independent of extracellular calcium and
apparently enhanced by the absence of extracellular calcium. These findings combined with previous data which showed that P R L release D A in the presence of tetrodotoxin [4], strongly suggest that P R L is exerting its effect directly upon dopaminergic terminals o f the CS without involving inflow of extracellular calcium ions or altering D A synthesis. It is possible that continuous exposure to P R L and the prolonged increase in basal D A release can deplete a hypothetical AMPH-sensitive D A storage pool sufficiently to result in an attenuated AMPH-stimulated D A response. However, the present results showed that postsuperfusion D A content levels were not affected by 1 0 - S M PRL. An alternate explanation is that P R L might interfere with the A M P H mediated D A release mechanism. Specifically, P R L might competitively interact with A M P H at D A reuptake sites. It is worth emphasizing that AMPH-induced D A release is a calcium-independent process as well [9] and thus further supports the above hypothesis. In summary, these data showed that P R L can release D A from the CS which is an extracellular calcium-independent process. Prolactin-induced attenuation of AMPH-induced D A release is probably not due to depletion of D A stores. The elusive nature of P R L binding sites in the rat striatum and the effects on D A release and behavior might be elucidated by examining interactions of P R L with the D A reuptake mechanism. This work was supported by N I H Grants PHS 5 732GM7143 to N.J.L., PHS 2 R01 HD14625 to V.D.R., and the Parkinson Disease Foundation and United Way of Stark County to D.E.D.
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