Neuroendocrinology 22: 206-215 (1976)
Selective Actions of Prolactin on Catecholamine Turnover in the Hypothalamus and on Serum LH and FSH1 G.A. G udelsky2, J. Simpkins3, G .P. M ueller3, J. M eites3 and K.E. M oore2 Departments of Pharmacology and Physiology, Michigan State University, East Lansing, Mich.
Key Words. Prolactin • Dopamine • Median eminence • Anterior hypothalamus • LH • FSH Abstract. The effects of prolactin (PRL) administration on catecholamine turnover in various brain regions of ovariectomized rats were determined by observing the decline of dopamine and norepinephrine concentrations after a-methyltyrosine (*MT) administra tion. PRL had no effect on the steady state concentration of dopamine in the median eminence, anterior hypothalamus and corpus striatum or on the norepinephrine concentra tion in the anterior hypothalamus. However, PRL selectively enhanced dopamine turnover in the median eminence and anterior hypothalamus after a latent period of 10-26 h. In addition, PRL administration significantly decreased serum concentrations of LH and FSH. These results suggest that the PRL-induced increase in activity of dopaminergic neurons in the median eminence or anterior hypothalamus may be responsible for the reduction of the post-castration rise in serum concentrations of LH and FSH.
The rat hypothalamus contains high concentrations of both dopamine and norepinephrine which are associated with specific neuronal pathways. Tuberoinfundibular neurons have dopamine-containing cell bodies in the arcuate and periventricular nuclei and terminals in the external layer of the 1 This work was aided in part by grants from the National Institute of Arthritis, Metabolism and Digestive Diseases (AM 04784) and from the National Cancer Institute (CA 10771) to J. Meites, and from the National Institute of Neurological Diseases and Stroke (NS 09174) to K.E. Moore. G.A. Gudelsky is a predoctoral student supported by USPHS training grant GM 1761. 2 Department of Pharmacology. 3 Department of Physiology.
Downloaded by: Nagoya University 133.6.82.173 - 6/19/2018 2:04:03 PM
Received: August 17th, 1976; accepted: October 2nd, 1976.
G udelsky/S impkins/M ueller/M eites/M oore
207
median eminence [F uxe, 1963; Bjorklund et al., 1973]. The dopamine content of the anterior hypothalamus appears to be associated with terminals of an incerto-hypothalamic pathway originating from cell bodies in the zona incerta [Bjorklund et al., 1975], while the norepinephrine in this area is present in axon terminals which arise from cell bodies in the brain stem [D ahlstrom and F uxe, 1964; U ngerstedt, 1971; J onsson et al., 1972], Hypothalamic monoamines have been postulated to mediate the secretion of anterior pituitary hormones, either by influencing the release of hypo thalamic peptide hormones or by acting directly on the anterior pituitary [F uxe and H okfelt, 1967; H5 kfelt and F uxe, 1972a; M c C ann et al., 1972; M eites et al., 1972], Tuberoinfundibular dopaminergic neurons appear to be responsible for tonic inhibition of prolactin (PRL) secretion [H okfelt and F uxe, 1972b; M eites et al., 1972; M acL eod, 1974], and also may release or inhibit the release of gonadotropins under some conditions [F uxe et al., 1976; Sawyer, 1975]. High circulating levels of PRL have been shown to inhibit PRL release by the in situ pituitary [M eites and C lemens, 1972; M acLeod, 1974] and may do so by increasing dopamine turnover in the median eminence [F uxe etal., 1969], There is some evidence that a 2nd func tional component for the regulation of PRL secretion in the female rat may be located in the anterior hypothalamus [see C hen and M eites, 1970; N eill, 1974], Therefore, the effects of PRL administration on the dynamics of dopamine and norepinephrine in the anterior hypothalamus are also of interest. The purpose of this study was to examine the effects of exogenous PRL administration on (a) dopamine and norepinephrine turnover in various brain regions of ovariectomized rats, as determined by depletion of cate cholamines after synthesis inhibition with «-methyltyrosine («MT), and on (b) serum concentrations of LH and FSH.
Female Sprague-Dawley rats (Spartan Research Animals, Inc., Haslett, Mich.) weighing 200-275 g each were ovariectomized and maintained in air-conditioned rooms under alternate 12 h periods of light and dark. The animals had free access to food and water. Rats were sacrificed 4 days after ovariectomy by decapitation between 09.00 and 13.00 h. Blood was collected and the serum was separated and stored at -20°C until assayed for LH and FSH by standard radioimmunoassays. The median eminence was removed from the hypothalamus with the aid of a dissecting microscope and fine scissors [C uello et at., 1974], The dissected tissue was composed primarily of the floor of the 3rd ventricle. The anterior hypothalamus was removed by making cuts rostral to and caudal to the optic
Downloaded by: Nagoya University 133.6.82.173 - 6/19/2018 2:04:03 PM
Materials and Methods
208
G udelsky/S impkins/M ueller/M eites/M oore
chiasm. The lateral borders were the hypothalamic sulci. The tissue samples from the anterior hypothalamus were approximately 1 x 4 x 3 rnm and the protein content was 172 ± 7 /ig. Bilateral samples of corpus striatum also were removed. Biochemical and radioimmunoassay procedures. The dopamine concentrations of median eminence and corpus striatum samples were determined by using a modification of the radiochemical enzymatic method of C uello elal. [1973], as described in detail by Moore and P hilupson [1975], Anterior hypothalamic samples were analyzed for dopamine and norepinephrine using a similar radiochemical enzymatic method [Coyle and H enry, 1973], Serum LH and FSH were measured by the N1AMDD RIA kits. Results are expressed in terms of the standards NIAMDD-LH-RP-1 and NIAMDD-FSH-RP-I, respectively. Protein was determined as described by Lowry et al. [1951]. PRL and drug administration. PRL (ovine, NIH-P-S11, obtained from the Pituitary Hormone Distribution Program) was dissolved in physiological saline at pH 11.0 and adjusted to pH 7.5 with 0.1 n NaOH. DL-a-methyltyrosine methyl ester HC1 (Regis Chemical Co., Morton Grove, III.) was dissolved in saline. Two groups of rats were given injections of PRL (5.0 mg/kg, s.c.) or vehicle every 8 h for 1 or 3 days and sacrificed 2 h after the last injection, or 26 and 74 h after the 1st injection, respectively. Another group was given PRL 10 and 2 h before decapitation; a 4th group was given PRL 2 h before decapitation. PRL- and vehicle-treated groups were divided so that half of each group received saline (zero time controls) or «MT (250 mg/kg, i.p.) 1 h before sacrifice. Student’s /-test was used to determine the significance of differences between the vehicle- and PRL-treated groups; the level of significance chosen was p < 0.05
Results
Effects o f PRL on txMT-induced Depletion of Brain Catecholamines Pretreatment with PRL had no effect on the steady state concentration of dopamine in the median eminence. That is, in saline-treated animals, the dopamine concentration in the median eminence of vehicle- and PRL-pretreated rats was the same. Therefore, these values were combined and means are represented as the horizontal lines in figure 1.
Selective Actions of Prolactin on Catecholamine Turnover in the Hypothalamus and on Serum LH and FSH1 G.A. G udelsky2, J. Simpkins3, G .P. M ueller3, J. M eites3 and K.E. M oore2 Departments of Pharmacology and Physiology, Michigan State University, East Lansing, Mich.
Key Words. Prolactin • Dopamine • Median eminence • Anterior hypothalamus • LH • FSH Abstract. The effects of prolactin (PRL) administration on catecholamine turnover in various brain regions of ovariectomized rats were determined by observing the decline of dopamine and norepinephrine concentrations after a-methyltyrosine (*MT) administra tion. PRL had no effect on the steady state concentration of dopamine in the median eminence, anterior hypothalamus and corpus striatum or on the norepinephrine concentra tion in the anterior hypothalamus. However, PRL selectively enhanced dopamine turnover in the median eminence and anterior hypothalamus after a latent period of 10-26 h. In addition, PRL administration significantly decreased serum concentrations of LH and FSH. These results suggest that the PRL-induced increase in activity of dopaminergic neurons in the median eminence or anterior hypothalamus may be responsible for the reduction of the post-castration rise in serum concentrations of LH and FSH.
The rat hypothalamus contains high concentrations of both dopamine and norepinephrine which are associated with specific neuronal pathways. Tuberoinfundibular neurons have dopamine-containing cell bodies in the arcuate and periventricular nuclei and terminals in the external layer of the 1 This work was aided in part by grants from the National Institute of Arthritis, Metabolism and Digestive Diseases (AM 04784) and from the National Cancer Institute (CA 10771) to J. Meites, and from the National Institute of Neurological Diseases and Stroke (NS 09174) to K.E. Moore. G.A. Gudelsky is a predoctoral student supported by USPHS training grant GM 1761. 2 Department of Pharmacology. 3 Department of Physiology.
Downloaded by: Nagoya University 133.6.82.173 - 6/19/2018 2:04:03 PM
Received: August 17th, 1976; accepted: October 2nd, 1976.
G udelsky/S impkins/M ueller/M eites/M oore
207
median eminence [F uxe, 1963; Bjorklund et al., 1973]. The dopamine content of the anterior hypothalamus appears to be associated with terminals of an incerto-hypothalamic pathway originating from cell bodies in the zona incerta [Bjorklund et al., 1975], while the norepinephrine in this area is present in axon terminals which arise from cell bodies in the brain stem [D ahlstrom and F uxe, 1964; U ngerstedt, 1971; J onsson et al., 1972], Hypothalamic monoamines have been postulated to mediate the secretion of anterior pituitary hormones, either by influencing the release of hypo thalamic peptide hormones or by acting directly on the anterior pituitary [F uxe and H okfelt, 1967; H5 kfelt and F uxe, 1972a; M c C ann et al., 1972; M eites et al., 1972], Tuberoinfundibular dopaminergic neurons appear to be responsible for tonic inhibition of prolactin (PRL) secretion [H okfelt and F uxe, 1972b; M eites et al., 1972; M acL eod, 1974], and also may release or inhibit the release of gonadotropins under some conditions [F uxe et al., 1976; Sawyer, 1975]. High circulating levels of PRL have been shown to inhibit PRL release by the in situ pituitary [M eites and C lemens, 1972; M acLeod, 1974] and may do so by increasing dopamine turnover in the median eminence [F uxe etal., 1969], There is some evidence that a 2nd func tional component for the regulation of PRL secretion in the female rat may be located in the anterior hypothalamus [see C hen and M eites, 1970; N eill, 1974], Therefore, the effects of PRL administration on the dynamics of dopamine and norepinephrine in the anterior hypothalamus are also of interest. The purpose of this study was to examine the effects of exogenous PRL administration on (a) dopamine and norepinephrine turnover in various brain regions of ovariectomized rats, as determined by depletion of cate cholamines after synthesis inhibition with «-methyltyrosine («MT), and on (b) serum concentrations of LH and FSH.
Female Sprague-Dawley rats (Spartan Research Animals, Inc., Haslett, Mich.) weighing 200-275 g each were ovariectomized and maintained in air-conditioned rooms under alternate 12 h periods of light and dark. The animals had free access to food and water. Rats were sacrificed 4 days after ovariectomy by decapitation between 09.00 and 13.00 h. Blood was collected and the serum was separated and stored at -20°C until assayed for LH and FSH by standard radioimmunoassays. The median eminence was removed from the hypothalamus with the aid of a dissecting microscope and fine scissors [C uello et at., 1974], The dissected tissue was composed primarily of the floor of the 3rd ventricle. The anterior hypothalamus was removed by making cuts rostral to and caudal to the optic
Downloaded by: Nagoya University 133.6.82.173 - 6/19/2018 2:04:03 PM
Materials and Methods
208
G udelsky/S impkins/M ueller/M eites/M oore
chiasm. The lateral borders were the hypothalamic sulci. The tissue samples from the anterior hypothalamus were approximately 1 x 4 x 3 rnm and the protein content was 172 ± 7 /ig. Bilateral samples of corpus striatum also were removed. Biochemical and radioimmunoassay procedures. The dopamine concentrations of median eminence and corpus striatum samples were determined by using a modification of the radiochemical enzymatic method of C uello elal. [1973], as described in detail by Moore and P hilupson [1975], Anterior hypothalamic samples were analyzed for dopamine and norepinephrine using a similar radiochemical enzymatic method [Coyle and H enry, 1973], Serum LH and FSH were measured by the N1AMDD RIA kits. Results are expressed in terms of the standards NIAMDD-LH-RP-1 and NIAMDD-FSH-RP-I, respectively. Protein was determined as described by Lowry et al. [1951]. PRL and drug administration. PRL (ovine, NIH-P-S11, obtained from the Pituitary Hormone Distribution Program) was dissolved in physiological saline at pH 11.0 and adjusted to pH 7.5 with 0.1 n NaOH. DL-a-methyltyrosine methyl ester HC1 (Regis Chemical Co., Morton Grove, III.) was dissolved in saline. Two groups of rats were given injections of PRL (5.0 mg/kg, s.c.) or vehicle every 8 h for 1 or 3 days and sacrificed 2 h after the last injection, or 26 and 74 h after the 1st injection, respectively. Another group was given PRL 10 and 2 h before decapitation; a 4th group was given PRL 2 h before decapitation. PRL- and vehicle-treated groups were divided so that half of each group received saline (zero time controls) or «MT (250 mg/kg, i.p.) 1 h before sacrifice. Student’s /-test was used to determine the significance of differences between the vehicle- and PRL-treated groups; the level of significance chosen was p < 0.05
Results
Effects o f PRL on txMT-induced Depletion of Brain Catecholamines Pretreatment with PRL had no effect on the steady state concentration of dopamine in the median eminence. That is, in saline-treated animals, the dopamine concentration in the median eminence of vehicle- and PRL-pretreated rats was the same. Therefore, these values were combined and means are represented as the horizontal lines in figure 1.
