Original Paper Neuroendocrinology 1992 ;56:889-894
Department of Physiology, Neuropeptide Division. The University of Texas Southwestern Medical Center at Dallas, Dallas, Tex., USA
The Effect of Galanin on Growth Hormone-Releasing Factor and Somatostatin Release from Median Eminence Fragments in vitro
Key Words
Abstract
Median eminence In vitro incubation Growth hormone-releasing factor Somatotrophin release-inhibiting factor
Galanin has been reported to stimulate secretion of GH in humans and rats. Thus, to inves tigate whether the effect of galanin on GH release is the result of either a stimulation of GH-releasing factor (GRF) and/or an inhibition of somatostatin (SRI F) release, we have evaluated the action of galanin on the release of SRIF and GRF from median eminence (ME) fragments in vitro. The MEs from adult male rats were incubated in Krebs-Ringer bicarbonate-glucose buffer, pH 7.4, at 37 °C, in an atmosphere of 95% Ch, 5% CO; with constant shaking for 30 min. Medium was discarded and replaced by medium containing various concentrations of galanin (I0~'°-10"7 M). Galanin stimulated SRIF and GRF re lease in a dose-related manner. This effect was significant at concentrations varying from 10~® to I0~7 M. To determine the mechanism by which galanin stimulated SRIF and GRF release, MEs were incubated with pimozide (dopaminergic blocker), phentolamine (a-adrenergic blocker) or naloxone (opioid blocker), at concentrations of I0~6 M, and the effect of galanin was then evaluated. Phentolamine and naloxone did not alter the stimulatory effect of galanin, but when galanin was tested with pimozide, the galanin-induced release of SRIF and GRF was blocked. To determine whether the effect of galanin is mediated through D-l and/or D-2 dopamine receptors, selective antagonists of D-l (SCH 23390) and D-2 receptors (domperidone) were used (10~7 M) in the presence of galanin (1(F7 M). The stimulatory effect of galanin on SRIF release was unaffected by domperidone, although SCH 23390 inhibited galanin-evoked SRIF release. However, galanin-evoked GRF release was completely abolished by either domperidone or SCH 23390. Since GRF stimulates SRIF release in vitro, we hypothesized that the SRIF-stimulating action of galanin might be mediated by GRF accumulating in the static incubation system; however GRF antiserum (1:100) failed to alter this effect. These results demonstrate that galanin stimulates GRF and SRIF from ME fragments in vitro by a dopaminergic mechanism. In vivo, this GRF acti vates release of GH from the somatotropes. Thus, the stimulation of GRF release by galanin will increase GH release. Since galanin stimulates GH release in vivo, its stimulatory effect on SRIF release via a dopaminergic mechanism was puzzling. Since this effect was not blocked by GRF antiserum, the mediation of the effect by accumulated GRF appears to be ruled out. Therefore, we hypothesize that the effect is mediated by galanin itself acting as a negative feedback, when present in high concentration intrahypothalamicaliy, to stimulate SRIF release. This effect would not be manifest in vivo except at very high secretion rates of galanin since the released galanin would enter the portal vessels instead of remaining in the tissue as in this static incubation system employed here
Received: October 18, 1991 Accepted after revision: May 27. 1992
M.C. Aguila Department o f Physiology. Neuropeptide Division The University of Texas Southwestern Medical Center at Dallas 5323 Harry Hines Boulevard Dallas,TX 75235-9040 (USA)
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M. Cecilia Aguila Umeko Marubayashi Samuel M. McCann
lanin increases GH release from the pituitary gland in vitro [10-14], but similar studies have shown no effect on GH secretion [14]. Galanin has been shown to stimulate GRF release in vivo [8, 9] and in vitro [ 15]. The mechanism by which galanin stimulates GH re lease has not been completely elucidated. To investigate whether the effect of galanin at the hypothalamic level is the result of either a stimulation of GRF and/or an inhi bition of somatostatin (SRIF) release, we have evaluated its action on the release ofSRIFand GRF from ME frag ments in vitro. A preliminary report of these experiments has been published [16].
Materials and Methods Experimental Animals Adult male Sprague-Dawley rats (200-250 g) were purchased from Harlan (Madison, Wise., USA). Animals were housed under controlled conditions of temperature (23-25 °C) and lighting (14 h of light. 10 h of darkness). Rat Chow and water were provided ad libitum.
The neuropeptide galanin is a 29-amino-acid peptide derived from a large prohormone [I], Galanin was iso lated from porcine intestine [2], and it is widely distrib uted in central and peripheral neurons of several mam malian species, including man and rats [3). In the rat, the highest brain concentrations are found in the hypothala mus, especially in the median eminence (ME) [4, 5]. A subpopulation of immunocytochemically identified ga lanin neurons in the arcuate nucleus send their axons to the ME. These same neurons produce, store and release a growth hormone-releasing factor (GRF)-like peptide [6],Furthermore, central (third ventricular) and i.v. ad ministration of galanin has been shown to increase the release of growth hormone (GH) in conscious rats [7-9]. The peptide increases plasma GH in humans [10] follow ing its i.v. injection. Besides, it has been found that ga-
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Radioimmunoassays SRIF was measured as previously described [19] utilizing an an tiserum kindly provided by Dr. Louis de Palatis [20], The antiserum is used at a dilution of 1:50,000, and the labelled SRIF is added 24 h later; this provided a sensitivity of 3.1 pg/tube. GRF was as sayed using an RIA developed in our laboratories and previously described [21], The antiserum (RAS 8068) was purchased from Peninsula Lab (Belmont, Calif., USA). It did not cross-react with human GRF-44-NH2, human GRF-40, porcine GRF, PHI or VIP. The lowest level of detection was 1.0 pg/tube and the highest 250 pg/tube.
Aguila/Marubayashi/McCann
Galanin-Induced Release ofSRIFand GRF
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Fig. 1. Effect of galanin on SRIF and GRF release from ME fragments in vitro. In this and subsequent figures, values are the mean ± SEM, and the number of determi nations is given above each point. **p < 0.001, *p < 0.05 vs. basal (B).
ME Incubations The procedure used has been previously reported [17, 18]. Briefly, after decapitation, the brain of the rat was removed and the ME was dissected free under a stereoscopic microscope. The tissue samples included only the ME and the proximal stump of the pitu itary stalk. Four MEs were incubated in 0.25 ml Krebs-Ringer bicarbonate glucose buffer (KRBG). pH 1A with bacitracin (2 x 10 ' A/), a peptidase inhibitor, in an atmosphere of 95% 02/5% CO; with con stant shaking (60 cycles/min) at 37 °C. At the end of 30 min of preincubation, the medium was discarded and replaced with fresh medium containing several concentrations of rat galanin (10"7- 10”11 M). Incubation was then continued for another 30 min. Pimozide and domperidone were previously dissolved in 0 .1 N tartaric acid. Phentolamine, naloxone and SCH 23390 were dis solved in water. At the moment of the experiment, 10"'’ M concen tration of the antagonists was prepared in KRBG and added to the ME fragments during the preincubation. Then, the medium was dis carded and exposed simultaneously to the inhibitors and 10”7 M concentration of galanin for an additional 30 min. Incubation media were stored at -70 °C prior to assay for SRIF and GRF.
Fig. 2. Effect of dopaminergic receptor blockade by pimozide (PIM) on galanin (GAL)-induced SR1F (a) and GRF (b) release from ME in vitro. **p < 0.001 vs. basal (B).
NAL
GAL GAL
from ME fragments. **p < 0.001, *p < 0.025 vs. basal (B).
+
B
NAL
GAL GAL
Fig. 3. Effect of opioid receptor blockade by naloxone (NAL) on galanin (GAL)-evoked SRIF (a) and GRF (b) release from ME tissue. **p < 0.001, *p < 0.05 vs. basal (B).
Fig. 5. Effect of D-1 DA receptor antagonist (SCH 2339; Sch) on galanin (GAL)-induced SRIF (a) and GRF (b) release from ME terminals in vitro. **p < 0.001 vs. basal (B).
Statistical Analysis Differences amongst several groups were analyzed by one-way analysis of variance followed by the Student-Newman-Keul multiple range comparison test for unequal replications.
Effects o f Dopaminergic. Adrenergic and Opioidergic Receptor Blockade on Galanin-Stimulated GRF and SRIF Release To determine if the effect of galanin on SRIF and GRF release was mediated by dopamine (DA), noradrenaline or opioid peptides, pimozide (dopaminergic receptor blocker), naloxone (opioid receptor blocker) and phen tolamine («-adrenergic receptor blocker) were added at a concentrations of 10~6 M to the medium that also con tained 10~7 M galanin (fig. 2-4). The receptor blockers were also incubated alone with ME tissue. The concentra tion of 10"7 M galanin was effective as before. Pimozide,
Results Effects o f Galanin on GRF and SRIF Releasefrom ME Tissue in vitro Galanin stimulated SRIF and GRF release in a doserelated manner (fig. 1), concentrations of 10" and 10~8 M significantly stimulated both, SRIF and GRF release.
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+
B
Fig. 4. Effect of «-adrenergic receptor blockade by phentolamine (PH E) on galanin (GAL)-stimulated SRIF (a) and GRF (b) release
stimulatory effect of galanin (10"7 M) on SRIF release was not altered by the GRF antibody (data not shown). The antibody at this concentration had been previously found effective to block IGF-I-induced SRIF release [22],
Discussion
Domp GAL Domp
B
Domp GAL Oomp
Fig. 6. Effect of the D-2 DA receptor antagonist, domperidone (Domp) on galanin (GAL)-evoked SRIF (a) and GRF (b) release from ME fragments. **p < 0.001 vs. basal (B).
phentolamine and naloxone by themselves had no signifi cant effect on GRF and SRIFrelease; however, the stimu latory effect of galanin on GRFand SRI F was inhibited in the presence of pimozide. Effect o f DA Receptor Blockade on Galanin-Induced SRIF and GRF Release To determine whether the effect of galanin is mediated through D-I and/or D-2 DA receptors, selective antago nists of D-l (SCH 23390) and D-2 receptors (domperi done) were used at a concentration of 10~6 M in the pres ence of 10"' M galanin (fig. 5, 6). SCH 23390 did not modify basal SRIF release, but it inhibited galaninevoked SRIF release (fig. 5a). However, basal GRF re lease was slightly reduced by SCH 23390 (fig. 5b). Like wise, the stimulatory effect of galanin on GRF release was completely blocked by SCH 23390 (fig. 5b). Domperi done did not modify basal SRIF on GRF release (fig. 6). The stimulatory effect of galanin on SRIF release was un affected by domperidone (fig. 6a). However, galaninevoked GRF release was completely abolished by dom peridone (fig. 6b). Effect o f Anti-Rat GRF on Galanin-Induced SRIF Release Since GRF stimulates SRIF release in vitro, we evalu ated the possibility that the effect of galanin on SRIF re lease is mediated by GRF release by incubating the MEs in the presence of a GRF antibody (RAS, 1:100). The
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B
The present study gives evidence for a neuroendo crine function of galanin in the normal male rat, and it shows that rat galanin has an effect to stimulate both GRF and SRIF release in a dose-related manner from isolated ME fragments. The effect of galanin was signif icant at concentrations of 10~x and 10"7 M, respectively, with respect to controls. The concentrations of galanin that are effective in this in vitro ME system are not dif ferent from those reported in anterior pituitary cell cul ture [13]. The effect of galanin was not altered by opioidergic or adrenergic receptor blockers (naloxone, phentolamine) at concentrations that have been pre viously shown to block their respective receptors in this system [23, 24]. In these studies, it appears that this in vitro response is not mediated by the release of norepi nephrine or opioid peptides from the ME. DA receptors of the CNS have been classified into two subtypes, based on their relationships to the effector enzyme adenylate cyclase. D-l receptors are linked in a stimulatory fashion to adenylate cyclase activity [25], while D-2 receptors are either unlinked or are inhibitory modulators of cAMP production [25]. The stimulatory ef fect of galanin on GRF and SRIF release was completely abolished by the DA receptor blocker pimozide. Pimo zide is a D-2 DA receptor blocker, but it is also a potent calmodulin blocker. Pimozide has an affinity for the D-2 receptors 2,500 times greater than it has for calmodulin [26] , Previous reports have shown a calmodulin depend ence of SRIF release from dispersed hypothalamic cells [27] and ME fragments [28]. Therefore, at a micromolar concentration it could have blocked galanin-stimulated SRIF release by either action. For these reasons, we tested domperidone, a selective D-2 DA receptor blocker [25]. The stimulatory effect of galanin on GRF release was completely blocked by domperidone, and the receptor blocker by itself decreased basal values of GRF release. Domperidone did not alter galanin-evoked SRIF release, suggesting that D-2 dopaminergic receptors are not in volved in the release of SRIF induced by galanin. Thus, the inhibitory effect of pimozide on galanin-induced SRIF release could have been caused by interference with the activation of calmodulin [26].
reports support our findings which indicate that galanin stimulates GRF release through the dopaminergic sys tem. The stimulatory effect of galanin on GRF has been earlier reported in vivo [9, 10], It has been suggested to be mediated by the adrenergic system [10]. That possibility was not supported in our experiments. It has been reported that galanin did not alter SRIF release from hypothalamic fragments [38], but changes in release were determined after 1 h of incubation. In 1 h many factors, such as degradation of the peptide or ultra shortloop feedbacks between SRIF, GRF and other fac tors, may be affecting the release. It was reported that DA increases SRIF release through D-2 receptors [23, 39]; however, under our experimental conditions, we found that galanin evoked SRIF release by a D-l receptor mech anism. It has also been described that galanin inhibited the K +-stimulated release of [3H] DA in ME fragments [5], The reason for such a discrepancy might be the condi tions (K+-stimulated vs. basal release) in which the ex periments were performed. Tire fact that anti-GRF serum did not alter the stimulatory effect of galanin on SRIF release indicates that GRF does not mediate galaninevoked SRIF release. The facts that galanin releases both GRF and SRIF and yet induces dramatic GH release following its third ventricular injection are puzzling. One possible explana tion of this apparent paradox is that the GRF and galanin are coreleased in vivo into the hypophyseal portal capil laries, since galanin and GRF have an additive effect in increasing GH release from rat pituitary cells [34], the in hibitory effect of coreleased SRIF on the somatotrophs may be blocked. Lastly, DA inhibits the release of galanin from both the basal hypothalmus [35] and the pituitary itself [35, 36]. Therefore, galanin-induced release of DA may act as a negative feedback to inhibit release of ga lanin from both the hypothalamus and the anterior pitu itary gland. Further studies need to be performed to clar ify how the mechanisms described in the present work operates in physiological in vivo conditions.
Acknowledgments This work was supported by NIH grants HD09988 (S.M.M.), DK10073 (S.M.M.). DK40994 (S.M.M.) and NS2682I (MCA). The helpful advice of Dr. L. Krulich is greatly appreciated. We also thank Judy Scott for her secretarial assistance.
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SCH 23390, the 3-methyl 7-chloro analog of 38393, a selective antagonist of the D-l dopaminergic receptor [29], completely blocked the effect of galanin on GRF re lease, and SCH 23390 by itself decreased basal values of GRF. The fact that basal GRF release was also reduced by the dopaminergic antagonist suggests that there was a tonic dopaminergic activity in the fragments that was par tially responsible for basal GRF release. The stimulatory effect of galanin on SRIF release was reduced by the D-l dopaminergic receptor blocker, which suggests that this effect was mediated by D -1 receptors. Although it is note worthy that the ME is full of dopaminergic nerve termi nals, the density of DA receptors in the ME is lowest among the CNS structures [30]. D-l and D-2 binding sites have been reported to be distributed throughout most of the hypothalamus [31]. On the other hand, very dense galanin fiber networks have been observed in the ME, especially in the external layer where they show a marked overlap with tyrosine hy droxylase immunoreactive (TH-IR) fibers [32], In addi tion, GRF-IR fibers in the external layer of the ME show a high degree of overlap with TH-IR fibers, suggesting a coexistence of DA, GRF and galanin in nerve endings around the portal vesssels [32], In addition, a subpopula tion of galanin neurons in the arcuate nucleus sending axons to the ME produces, stores and releases a GRFlike peptide [6]. Furthermore, DA, L-dopa and a variety of DA agonists have been reported to cause GH release and increase plasma concentrations of the hormone [33, 34], Intraventricular infusions of DA and DA agonists, as well as peripheral administration of a DA-releasing agent also elevate plasma GH levels [34], DA increased GRF release in hypothalamic fragments perfused in vitro in the presence of SRIF antiserum [35], Likewise, in a different study, we found a direct effect of DA on GRF release from hypothalamic fragments incubated in vitro [unpubl. observations]. Although the functions of the D-l receptors are not fully understood, there is considerable evidence that their major function in the CNS is in the regulation of the ex pression of the activity of the D-2 receptors. That is, a decrease of the D-l activity (or blockade with SCH) im pairs the manifestations of the activity of the D-2 recep tors and, conversely, their increased activity augments them [36, 37]. It is possible that SCH did not block a D-l receptor which directly mediates the effect of DA released by galanin on SRIF or GRF, but prevented the effect of the activation of the D-2 receptor. In this way, it is possible to understand how galanin evoked GRF release by a D-l and a D-2 receptor mechanism. These previous
1 Rokaeus A, Brownstein MJ : Construction of a porcine adrenal medullary cDNA library and nucleotide sequence analysis of two clones en coding a galanin precursor. Proc Natl Acad Sei USA 1986:83:6287-6291. 2 Tatemoto K, Rokaeus A, Fornvall H, McDo nald TF, Mutt V: Galanin - A novel biologi cally active neuropeptide. FEBS Lett 1983; 164:124-128. 3 Rokaeus A: Galanin: A newly isolated bio logically active neuropeptide. Trends Neurosci 1987;10:158-164. 4 Chang JL, Christofides NA, Anand P, Gibson SJ, Allen YS. Su HC, Tatemoto K, Morrison JF. Polak JM, Blood SR: Distribution of ga lanin immunoreactivity in the central nervous system and the response of galanin containing neuronal pathways to injury. Neuroscience 1985;16:343-354. 5 Nordstrom O, Melander T, Hökfelt T, Bartfai T, Golstein M: Evidence for an inhibitory ef fect of the peptide galanin on dopamine re lease from rat median eminence. Neurosci Lett 1987:73:21-26. 6 Niimi M, Takahara J. Sato M, Kawanishi K: Immunohistochemical identification of ga lanin and growth-hormone-releasing-factorcontaining neurons projecting to the median eminence of the rat. Neuroendocrinology 1990;51:572-575. 7 Ottlecz A. Samson WK. McCann SM: Ga lanin: Evidence for a hypothalamic site of action to release growth hormone. Peptides 1986;7:51-53. 8 Murakami Y, Kato Y, Koshiyama H, Inoue T, Yanaihara N: Galanin stimulates growth hor mone (GH) secretion via GH-releasing factor (GRF) in conscious rat. Eur J Pharmacol 1987;136:415-418. 9 Murakami Y, Kalo Y, Shimatsu A, Koshiyama H, Haltori N, Yanaihara N, Imura H: Possible mechanisms involved in growth hormone se cretion induced by galanin in the rat. Endocri nology 1989;124:1224-1229. 10 Bauer FE, Ginsberg L, Venetikou M, MacKay DJ, Burrin JM, Bloom SR: Growth-hormone release in man induced by galanin, a new hy pothalamic peptide. Lancet 1986:ii: 192—195. 11 Ottlecz A, SnyderGD, McCann SM: Regula tory role of galanin in control of hypotha lamic-anterior pituitary function. Proc Natl Acad Sei USA 1988;85:9861-9865. 12 Gabriel SM. Milbury CM, Nathanson JA, Martin JB: Galanin stimulates rat pituitary growth hormone secretion in vitro. Life Sei 1988;42:1981-1986. 13 Torsello A, Sellan R, Cella SG, Locatelli V, Müller EE: Age-dependent modulation by galanin of growth hormone release from rat pituitary cells in culture. Life Sei I990;47:I86I- 1866. 14 Sato M. Takahara J, Niimi M, Tagawa R. Shozo I: Characterization of the stimulatory effect of galanin on growth hormone release from the rat anterior pituitary. Life Sei 1991; 48:1639-1644.
15 Kitajima N. Chihara K, Abe H. Okimura Y, Shakutsui S: Galanin stimulates immunoreactive growth hormone releasing factor secretion from rat hypothalamic slices perifused in vitro. LifeSci 1990;47:2371-2376. 16 McCann SM, Reznikov AG, Aguila MC, Marubayashi U, Gutkowska J, Rettori V: Role of galanin in control of hypothalamic pituitary function; in Hokfell T, Bartfai T (eds): Wenner-Gren Symposium. Stockholm, MacMillian Press, 1991, pp 307-319. 17 Ojeda SR. Negro-Vilar A. McCann SM: Catecholaminergic modulation of LHRH release by median eminence terminals: In vitro stud ies. Endocrinology 1979;104:1749-1757. 18 Aguila MC, McCann SM: Stimulation of so matostatin release in vitro by a synthetic human growth hormone-releasing factor by a nondopaminergic mechanism. Endocrinology 1985:117:762-765. 19 Arimura A, Sato H, Coy DH, Schally AV: Ra dioimmunoassay GH-release inhibiting hor mones. Proc Soc Exp Biol Med 1976:148: 784-789. 20 De Palalis LR. Khorram O, McCann SM: Dy namic changes in pituitary and plasma GH in the hypothalamic content of immunoreaclive somatostatin throughout pregnancy in the rat (abstract # 1329). 64th Annu Meet of the EndocrSocSan Francisco, June 1982. 21 Aguila MC, Pickle RL, Yu WH. McCann SM: Roles of somatostatin and growth hormone releasing factor in ether stress inhibition of growth hormone release. Neuroendocrinology 1991;54:515-520. 22 Aguila MC: Insulin-like growth factor I (1GFI) regulates somatostatin (SR1F) release and messenger ribonucleic acid levels in rat hypo thalamus (abstract 1283). 73rd Annu Meet of the Endocr Soc, Washington, D.C., June 1991. 23 Negro-Vilar A. Ojeda SR, Arimura A. McCann SM: Dopamine and norepinephrine stimulate somatostatin release by median emi nence fragments in vitro. Life Sci 1978:23: 1493-1497. 24 Aguila MC, McCann SM: Evidence that growth hormone-releasing factor stimulates somatostatin release in vitro via |)-endorphin. Endocrinology 1988;120:341-344. 25 Stoof JC. Kebabian JW: Two dopamine recep tors: Biochemistry, physiology and pharma cology. LifeSci 1984;35:2281-2296. 26 Weiss B, Wallace TL: Mechanisms and phar macological implications of altering calmo dulin activity; in Cheung WY (ed): Calmo dulin, Calcium and Cell Function. London, Academic Press, 1984, vol I, pp 102-164. 27 Drouva SV, Epelbaum J, Laplante E, Kordon C: Calmodulin involvement on the Ca* +-de pendent release of LHRH and SRIF in vitro. Neuroendocrinology 1984;38:189-192. 28 Aguila MC, McCann SM: Calmodulin de pendence of somatostatin (SRIF) release stim ulated by growth hormone-releasing factor (GRF). Endocrinology 1988;123:305-309.
29 Itoh Y. Beaulieu M, Kebabian JW: The chemi cal basis for the blockade of the D-l dopa mine receptor by SCH 23390. Eur J Pharma col 1984:100:119-122. 30 Dubois A: Autoradiographic distribution of the Di, agonist [’H] SKF 38393 in the rat brain and spinal cord. Comparison with the distribution of D: receptors. Neuroscience 1986;19:125-137. 31 Mansour A, Meador-Wooddruff JH. Bunzow JR, Civelli O, Akil H, Watson SJ: Localization or dopamine D; receptor mRNA and Di and D; receptor binding in the rat brain and pitu itary: An in situ hybridization-receptor auto radiographic analysis. J Neurosci 1990:10: 2587-2600. 32 Everitt BJ, Meister B, Hokfelt T, Melander T, Terenius L, Rokaeus A, Theodorsson-Norheim E, Dockray G, Edwarson J, Cucllo C, Eide R. Goldstein M, Hemmings H, Ouimet CH. Walaas I, Greengard P. Vale W. Weber E, Wu JY. Chang KJ: The hypothalamic arcuate nucleus-median eminence complex: Immunohistochemistry of transmitters, peptides and DARPP-32 with special reference to coexistence in dopamine neurons. Brain Res [Brain Res Rev] 1986;11:97-155. 33 Müller EE: Neural control of somatotropic function. Physiol Rev 1987;67:962-1053. 34 Vijayan E, Krulich L. McCann SM: Catecholaminergic regulation of TSH and growth hor mone release in ovaricctomized and steroid primed rats. Neuroendocrinology 1978:26: 174-185. 35 Kitajima N, Chihara K, Abe H, Okimura Y, Fujii Y, Sato M. Shakutsui S, Watanabe M, Fujita T: Effects of dopamine on immunoreactive growth hormone releasing factor and so matostatin secretion from rat hypothalamic slices perifused in vitro. Endocrinology 1989;124:69-76. 36 Waddington JL: Behavioral correlates of the action of selective D-l dopamine receptor antagonists. Biochem Pharmacol I986;35: 3661-3667. 37 Carlson JH. Bergstrom DA, Walters JR: Neu rophysiological evidence that D -1 dopamine receptor blockade attenuates postsynaptic but not autoreceptor-mediated effects of dopa mine agonists. EurJ Pharmacol 1986:23:237251. 38 Hulting AL, Meister B, Carlsson L, Hilding A, lsaksson O: On the role of the peptide ga lanin in regulation of growth hormone secre tion. Acta Endocrinol (Copenh) 1991 ; 125: 518-525. 39 Junicr MP, Dray F, Blair I, Capdevilla J, Dishman E, Falck JR. Ojeda SR: Epoxygenase products of arachidonic acid are endoge nous constituents of the hypothalamus in volved in D; receptor-mediated dopamine-in duced release of somatostatin. Endocrinology 1990; 126:1534-1540.
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References
Department of Physiology, Neuropeptide Division. The University of Texas Southwestern Medical Center at Dallas, Dallas, Tex., USA
The Effect of Galanin on Growth Hormone-Releasing Factor and Somatostatin Release from Median Eminence Fragments in vitro
Key Words
Abstract
Median eminence In vitro incubation Growth hormone-releasing factor Somatotrophin release-inhibiting factor
Galanin has been reported to stimulate secretion of GH in humans and rats. Thus, to inves tigate whether the effect of galanin on GH release is the result of either a stimulation of GH-releasing factor (GRF) and/or an inhibition of somatostatin (SRI F) release, we have evaluated the action of galanin on the release of SRIF and GRF from median eminence (ME) fragments in vitro. The MEs from adult male rats were incubated in Krebs-Ringer bicarbonate-glucose buffer, pH 7.4, at 37 °C, in an atmosphere of 95% Ch, 5% CO; with constant shaking for 30 min. Medium was discarded and replaced by medium containing various concentrations of galanin (I0~'°-10"7 M). Galanin stimulated SRIF and GRF re lease in a dose-related manner. This effect was significant at concentrations varying from 10~® to I0~7 M. To determine the mechanism by which galanin stimulated SRIF and GRF release, MEs were incubated with pimozide (dopaminergic blocker), phentolamine (a-adrenergic blocker) or naloxone (opioid blocker), at concentrations of I0~6 M, and the effect of galanin was then evaluated. Phentolamine and naloxone did not alter the stimulatory effect of galanin, but when galanin was tested with pimozide, the galanin-induced release of SRIF and GRF was blocked. To determine whether the effect of galanin is mediated through D-l and/or D-2 dopamine receptors, selective antagonists of D-l (SCH 23390) and D-2 receptors (domperidone) were used (10~7 M) in the presence of galanin (1(F7 M). The stimulatory effect of galanin on SRIF release was unaffected by domperidone, although SCH 23390 inhibited galanin-evoked SRIF release. However, galanin-evoked GRF release was completely abolished by either domperidone or SCH 23390. Since GRF stimulates SRIF release in vitro, we hypothesized that the SRIF-stimulating action of galanin might be mediated by GRF accumulating in the static incubation system; however GRF antiserum (1:100) failed to alter this effect. These results demonstrate that galanin stimulates GRF and SRIF from ME fragments in vitro by a dopaminergic mechanism. In vivo, this GRF acti vates release of GH from the somatotropes. Thus, the stimulation of GRF release by galanin will increase GH release. Since galanin stimulates GH release in vivo, its stimulatory effect on SRIF release via a dopaminergic mechanism was puzzling. Since this effect was not blocked by GRF antiserum, the mediation of the effect by accumulated GRF appears to be ruled out. Therefore, we hypothesize that the effect is mediated by galanin itself acting as a negative feedback, when present in high concentration intrahypothalamicaliy, to stimulate SRIF release. This effect would not be manifest in vivo except at very high secretion rates of galanin since the released galanin would enter the portal vessels instead of remaining in the tissue as in this static incubation system employed here
Received: October 18, 1991 Accepted after revision: May 27. 1992
M.C. Aguila Department o f Physiology. Neuropeptide Division The University of Texas Southwestern Medical Center at Dallas 5323 Harry Hines Boulevard Dallas,TX 75235-9040 (USA)
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M. Cecilia Aguila Umeko Marubayashi Samuel M. McCann
lanin increases GH release from the pituitary gland in vitro [10-14], but similar studies have shown no effect on GH secretion [14]. Galanin has been shown to stimulate GRF release in vivo [8, 9] and in vitro [ 15]. The mechanism by which galanin stimulates GH re lease has not been completely elucidated. To investigate whether the effect of galanin at the hypothalamic level is the result of either a stimulation of GRF and/or an inhi bition of somatostatin (SRIF) release, we have evaluated its action on the release ofSRIFand GRF from ME frag ments in vitro. A preliminary report of these experiments has been published [16].
Materials and Methods Experimental Animals Adult male Sprague-Dawley rats (200-250 g) were purchased from Harlan (Madison, Wise., USA). Animals were housed under controlled conditions of temperature (23-25 °C) and lighting (14 h of light. 10 h of darkness). Rat Chow and water were provided ad libitum.
The neuropeptide galanin is a 29-amino-acid peptide derived from a large prohormone [I], Galanin was iso lated from porcine intestine [2], and it is widely distrib uted in central and peripheral neurons of several mam malian species, including man and rats [3). In the rat, the highest brain concentrations are found in the hypothala mus, especially in the median eminence (ME) [4, 5]. A subpopulation of immunocytochemically identified ga lanin neurons in the arcuate nucleus send their axons to the ME. These same neurons produce, store and release a growth hormone-releasing factor (GRF)-like peptide [6],Furthermore, central (third ventricular) and i.v. ad ministration of galanin has been shown to increase the release of growth hormone (GH) in conscious rats [7-9]. The peptide increases plasma GH in humans [10] follow ing its i.v. injection. Besides, it has been found that ga-
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Radioimmunoassays SRIF was measured as previously described [19] utilizing an an tiserum kindly provided by Dr. Louis de Palatis [20], The antiserum is used at a dilution of 1:50,000, and the labelled SRIF is added 24 h later; this provided a sensitivity of 3.1 pg/tube. GRF was as sayed using an RIA developed in our laboratories and previously described [21], The antiserum (RAS 8068) was purchased from Peninsula Lab (Belmont, Calif., USA). It did not cross-react with human GRF-44-NH2, human GRF-40, porcine GRF, PHI or VIP. The lowest level of detection was 1.0 pg/tube and the highest 250 pg/tube.
Aguila/Marubayashi/McCann
Galanin-Induced Release ofSRIFand GRF
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Fig. 1. Effect of galanin on SRIF and GRF release from ME fragments in vitro. In this and subsequent figures, values are the mean ± SEM, and the number of determi nations is given above each point. **p < 0.001, *p < 0.05 vs. basal (B).
ME Incubations The procedure used has been previously reported [17, 18]. Briefly, after decapitation, the brain of the rat was removed and the ME was dissected free under a stereoscopic microscope. The tissue samples included only the ME and the proximal stump of the pitu itary stalk. Four MEs were incubated in 0.25 ml Krebs-Ringer bicarbonate glucose buffer (KRBG). pH 1A with bacitracin (2 x 10 ' A/), a peptidase inhibitor, in an atmosphere of 95% 02/5% CO; with con stant shaking (60 cycles/min) at 37 °C. At the end of 30 min of preincubation, the medium was discarded and replaced with fresh medium containing several concentrations of rat galanin (10"7- 10”11 M). Incubation was then continued for another 30 min. Pimozide and domperidone were previously dissolved in 0 .1 N tartaric acid. Phentolamine, naloxone and SCH 23390 were dis solved in water. At the moment of the experiment, 10"'’ M concen tration of the antagonists was prepared in KRBG and added to the ME fragments during the preincubation. Then, the medium was dis carded and exposed simultaneously to the inhibitors and 10”7 M concentration of galanin for an additional 30 min. Incubation media were stored at -70 °C prior to assay for SRIF and GRF.
Fig. 2. Effect of dopaminergic receptor blockade by pimozide (PIM) on galanin (GAL)-induced SR1F (a) and GRF (b) release from ME in vitro. **p < 0.001 vs. basal (B).
NAL
GAL GAL
from ME fragments. **p < 0.001, *p < 0.025 vs. basal (B).
+
B
NAL
GAL GAL
Fig. 3. Effect of opioid receptor blockade by naloxone (NAL) on galanin (GAL)-evoked SRIF (a) and GRF (b) release from ME tissue. **p < 0.001, *p < 0.05 vs. basal (B).
Fig. 5. Effect of D-1 DA receptor antagonist (SCH 2339; Sch) on galanin (GAL)-induced SRIF (a) and GRF (b) release from ME terminals in vitro. **p < 0.001 vs. basal (B).
Statistical Analysis Differences amongst several groups were analyzed by one-way analysis of variance followed by the Student-Newman-Keul multiple range comparison test for unequal replications.
Effects o f Dopaminergic. Adrenergic and Opioidergic Receptor Blockade on Galanin-Stimulated GRF and SRIF Release To determine if the effect of galanin on SRIF and GRF release was mediated by dopamine (DA), noradrenaline or opioid peptides, pimozide (dopaminergic receptor blocker), naloxone (opioid receptor blocker) and phen tolamine («-adrenergic receptor blocker) were added at a concentrations of 10~6 M to the medium that also con tained 10~7 M galanin (fig. 2-4). The receptor blockers were also incubated alone with ME tissue. The concentra tion of 10"7 M galanin was effective as before. Pimozide,
Results Effects o f Galanin on GRF and SRIF Releasefrom ME Tissue in vitro Galanin stimulated SRIF and GRF release in a doserelated manner (fig. 1), concentrations of 10" and 10~8 M significantly stimulated both, SRIF and GRF release.
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+
B
Fig. 4. Effect of «-adrenergic receptor blockade by phentolamine (PH E) on galanin (GAL)-stimulated SRIF (a) and GRF (b) release
stimulatory effect of galanin (10"7 M) on SRIF release was not altered by the GRF antibody (data not shown). The antibody at this concentration had been previously found effective to block IGF-I-induced SRIF release [22],
Discussion
Domp GAL Domp
B
Domp GAL Oomp
Fig. 6. Effect of the D-2 DA receptor antagonist, domperidone (Domp) on galanin (GAL)-evoked SRIF (a) and GRF (b) release from ME fragments. **p < 0.001 vs. basal (B).
phentolamine and naloxone by themselves had no signifi cant effect on GRF and SRIFrelease; however, the stimu latory effect of galanin on GRFand SRI F was inhibited in the presence of pimozide. Effect o f DA Receptor Blockade on Galanin-Induced SRIF and GRF Release To determine whether the effect of galanin is mediated through D-I and/or D-2 DA receptors, selective antago nists of D-l (SCH 23390) and D-2 receptors (domperi done) were used at a concentration of 10~6 M in the pres ence of 10"' M galanin (fig. 5, 6). SCH 23390 did not modify basal SRIF release, but it inhibited galaninevoked SRIF release (fig. 5a). However, basal GRF re lease was slightly reduced by SCH 23390 (fig. 5b). Like wise, the stimulatory effect of galanin on GRF release was completely blocked by SCH 23390 (fig. 5b). Domperi done did not modify basal SRIF on GRF release (fig. 6). The stimulatory effect of galanin on SRIF release was un affected by domperidone (fig. 6a). However, galaninevoked GRF release was completely abolished by dom peridone (fig. 6b). Effect o f Anti-Rat GRF on Galanin-Induced SRIF Release Since GRF stimulates SRIF release in vitro, we evalu ated the possibility that the effect of galanin on SRIF re lease is mediated by GRF release by incubating the MEs in the presence of a GRF antibody (RAS, 1:100). The
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B
The present study gives evidence for a neuroendo crine function of galanin in the normal male rat, and it shows that rat galanin has an effect to stimulate both GRF and SRIF release in a dose-related manner from isolated ME fragments. The effect of galanin was signif icant at concentrations of 10~x and 10"7 M, respectively, with respect to controls. The concentrations of galanin that are effective in this in vitro ME system are not dif ferent from those reported in anterior pituitary cell cul ture [13]. The effect of galanin was not altered by opioidergic or adrenergic receptor blockers (naloxone, phentolamine) at concentrations that have been pre viously shown to block their respective receptors in this system [23, 24]. In these studies, it appears that this in vitro response is not mediated by the release of norepi nephrine or opioid peptides from the ME. DA receptors of the CNS have been classified into two subtypes, based on their relationships to the effector enzyme adenylate cyclase. D-l receptors are linked in a stimulatory fashion to adenylate cyclase activity [25], while D-2 receptors are either unlinked or are inhibitory modulators of cAMP production [25]. The stimulatory ef fect of galanin on GRF and SRIF release was completely abolished by the DA receptor blocker pimozide. Pimo zide is a D-2 DA receptor blocker, but it is also a potent calmodulin blocker. Pimozide has an affinity for the D-2 receptors 2,500 times greater than it has for calmodulin [26] , Previous reports have shown a calmodulin depend ence of SRIF release from dispersed hypothalamic cells [27] and ME fragments [28]. Therefore, at a micromolar concentration it could have blocked galanin-stimulated SRIF release by either action. For these reasons, we tested domperidone, a selective D-2 DA receptor blocker [25]. The stimulatory effect of galanin on GRF release was completely blocked by domperidone, and the receptor blocker by itself decreased basal values of GRF release. Domperidone did not alter galanin-evoked SRIF release, suggesting that D-2 dopaminergic receptors are not in volved in the release of SRIF induced by galanin. Thus, the inhibitory effect of pimozide on galanin-induced SRIF release could have been caused by interference with the activation of calmodulin [26].
reports support our findings which indicate that galanin stimulates GRF release through the dopaminergic sys tem. The stimulatory effect of galanin on GRF has been earlier reported in vivo [9, 10], It has been suggested to be mediated by the adrenergic system [10]. That possibility was not supported in our experiments. It has been reported that galanin did not alter SRIF release from hypothalamic fragments [38], but changes in release were determined after 1 h of incubation. In 1 h many factors, such as degradation of the peptide or ultra shortloop feedbacks between SRIF, GRF and other fac tors, may be affecting the release. It was reported that DA increases SRIF release through D-2 receptors [23, 39]; however, under our experimental conditions, we found that galanin evoked SRIF release by a D-l receptor mech anism. It has also been described that galanin inhibited the K +-stimulated release of [3H] DA in ME fragments [5], The reason for such a discrepancy might be the condi tions (K+-stimulated vs. basal release) in which the ex periments were performed. Tire fact that anti-GRF serum did not alter the stimulatory effect of galanin on SRIF release indicates that GRF does not mediate galaninevoked SRIF release. The facts that galanin releases both GRF and SRIF and yet induces dramatic GH release following its third ventricular injection are puzzling. One possible explana tion of this apparent paradox is that the GRF and galanin are coreleased in vivo into the hypophyseal portal capil laries, since galanin and GRF have an additive effect in increasing GH release from rat pituitary cells [34], the in hibitory effect of coreleased SRIF on the somatotrophs may be blocked. Lastly, DA inhibits the release of galanin from both the basal hypothalmus [35] and the pituitary itself [35, 36]. Therefore, galanin-induced release of DA may act as a negative feedback to inhibit release of ga lanin from both the hypothalamus and the anterior pitu itary gland. Further studies need to be performed to clar ify how the mechanisms described in the present work operates in physiological in vivo conditions.
Acknowledgments This work was supported by NIH grants HD09988 (S.M.M.), DK10073 (S.M.M.). DK40994 (S.M.M.) and NS2682I (MCA). The helpful advice of Dr. L. Krulich is greatly appreciated. We also thank Judy Scott for her secretarial assistance.
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SCH 23390, the 3-methyl 7-chloro analog of 38393, a selective antagonist of the D-l dopaminergic receptor [29], completely blocked the effect of galanin on GRF re lease, and SCH 23390 by itself decreased basal values of GRF. The fact that basal GRF release was also reduced by the dopaminergic antagonist suggests that there was a tonic dopaminergic activity in the fragments that was par tially responsible for basal GRF release. The stimulatory effect of galanin on SRIF release was reduced by the D-l dopaminergic receptor blocker, which suggests that this effect was mediated by D -1 receptors. Although it is note worthy that the ME is full of dopaminergic nerve termi nals, the density of DA receptors in the ME is lowest among the CNS structures [30]. D-l and D-2 binding sites have been reported to be distributed throughout most of the hypothalamus [31]. On the other hand, very dense galanin fiber networks have been observed in the ME, especially in the external layer where they show a marked overlap with tyrosine hy droxylase immunoreactive (TH-IR) fibers [32], In addi tion, GRF-IR fibers in the external layer of the ME show a high degree of overlap with TH-IR fibers, suggesting a coexistence of DA, GRF and galanin in nerve endings around the portal vesssels [32], In addition, a subpopula tion of galanin neurons in the arcuate nucleus sending axons to the ME produces, stores and releases a GRFlike peptide [6]. Furthermore, DA, L-dopa and a variety of DA agonists have been reported to cause GH release and increase plasma concentrations of the hormone [33, 34], Intraventricular infusions of DA and DA agonists, as well as peripheral administration of a DA-releasing agent also elevate plasma GH levels [34], DA increased GRF release in hypothalamic fragments perfused in vitro in the presence of SRIF antiserum [35], Likewise, in a different study, we found a direct effect of DA on GRF release from hypothalamic fragments incubated in vitro [unpubl. observations]. Although the functions of the D-l receptors are not fully understood, there is considerable evidence that their major function in the CNS is in the regulation of the ex pression of the activity of the D-2 receptors. That is, a decrease of the D-l activity (or blockade with SCH) im pairs the manifestations of the activity of the D-2 recep tors and, conversely, their increased activity augments them [36, 37]. It is possible that SCH did not block a D-l receptor which directly mediates the effect of DA released by galanin on SRIF or GRF, but prevented the effect of the activation of the D-2 receptor. In this way, it is possible to understand how galanin evoked GRF release by a D-l and a D-2 receptor mechanism. These previous
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References
