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Neuropeptide Y modulates growth hormone but not luteinizing hormone secretion from prepuberal gilt anterior pituitary cells in culture
Domestic Animal Endocrinology 29 (2005) 548–555
Neuropeptide Y modulates growth hormone but not luteinizing hormone secretion from prepuberal gilt anterior pituitary cells in culture C.R. Barb ∗ , J.B. Barrett 1 USDA-ARS, Animal Physiology Research Unit, Russell Agricultural Research Center, P.O. Box 5677, Athens, GA 30604-5677, USA Received 1 December 2004; received in revised form 1 March 2005; accepted 16 March 2005
Abstract Pituitary cells, from seven 160- to 170-day-old pigs, were studied in primary culture to determine the affects NPY on LH and GH secretion at the level of the pituitary. On day 4 of culture, medium was discarded, plates were rinsed twice with serum-free medium and cells were cultured in 1 ml fresh medium without serum and challenged individually with 10−10 , 10−8 or 10−6 M [Ala15 ]-h growth hormone-releasing factor-(1–29)NH2 (GRF); 10−9 , 10−8 or 10−7 M GnRH or 10−9 , 10−8 , 10−7 or 10−6 M NPY individually or in combinations with 10−9 or 10−8 M GnRH or 10−8 or 10−6 M GRF. Cells were exposed to treatment for 4 h at which time medium was harvested and quantified for LH and GH. Basal LH secretion (control; n = 7 pituitaries) was 12 ± 6 ng/well. Relative to control at 4 h, 10−9 , 10−8 and 10−7 M GnRH increased (P < 0.01) LH secretion by 169, 176 and 197%, respectively. Neuropeptide-Y did not alter (P > 0.4) basal LH secretion nor 10−8 M GnRH-induced increase in LH secretion but 10−9 M GnRH-stimulated LH secretion was reduced by NPY and was not different from control or GnRH alone. Basal GH secretion (control; n = 7 pituitaries) was 56 ± 12 ng/well. Relative to control at 4 h, 10−10 , 10−8 and 10−6 M GRF increased GH secretion by 111%, 125% (P < 0.01) and 150% (P < 0.01), respectively. Only 10−6 M (134%) and 10−7 M (125%) NPY increased (P < 0.04) basal GH secretion. Addition of 10−9 , 10−8 and 10−7 M NPY in combination with 10−8 M GRF suppressed (P < 0.04) GRF-stimulated GH secretion. However, 10−9 M NPY enhanced (P < 0.06) the ∗
Corresponding author. Tel.: +1 706 583 8276. E-mail address: [email protected] (C.R. Barb). 1 Present address: USDA-ARS, Antimicrobial Resistance Research Unit, Russell Research Center, P.O. Box 5677, Athens, GA 30604-5677, USA. 0739-7240/$ – see front matter. Published by Elsevier Inc. doi:10.1016/j.domaniend.2005.03.004
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GH response to 10−6 M GRF. These results demonstrate that NPY may directly modulate GH secretion at the level of the pituitary gland. Published by Elsevier Inc. Keywords: Neuropeptide Y; Pituitary; Luteinizing hormone; Growth hormone; Pig
1. Introduction Neuropeptide-Y (NPY) is a potent regulator of feeding, energy expenditure and fat storage [1,2]. Furthermore, hypothalamic NPY is responsive to changes in energy balance and blood leptin concentrations [3–5]. Intracerebroventricular (ICV) administration of NPY suppressed luteinizing hormone (LH) secretion and increased growth hormone (GH) secretion and feed intake in the prepuberal gilt [6,7]. Moreover, in the prepuberal gilt feed restriction delayed the onset of puberty [8], increased GH levels [9] and suppressed leptin secretion [10]. Immunoreactive NPY cell bodies and fibers are located within the arcuate nuclei, ventromedial hypothalamus and median eminence [11]. The presence of NPY in the anterior pituitary in the pig [12] would suggest that hypothalamic NPY is transported via the hypophyseal portal blood circulation to the anterior pituitary. In support of this idea, NPY has been detected in the hypophyseal portal blood in the rat [13,14]. The above reports are consistent with a direct effect of NPY on pituitary hormone secretion. Thus, NPY may relay metabolic information to the brain–pituitary axis to regulate pituitary hormone secretion. Therefore, the present study was conducted to test the hypothesis that NPY acts directly on the anterior pituitary to modulate LH and GH secretion in the prepuberal gilt. 2. Materials and methods Pituitary glands were aseptically removed from seven intact prepuberal gilts 160 to 170 days of age. Ovaries were examined at slaughter and gilts were considered prepuberal because their ovaries were devoid of corpora albicantia and corpora lutea. All subsequent procedures were performed under sterile conditions. The anterior lobe was dissected from each pituitary gland; cells enzymatically dispersed and cultured as previously described [15]. Cells from each pig were cultured separately. Cell viability and number were assessed by counting cells which excluded trypan blue on a hemocytometer. Cell viability was 96%. Cells were diluted and plated, based on the number of live cells. Cells were plated 105 cells/ml with culture medium in a 24-well cluster plate. Culture medium was changed on day 3 (day of seeding = day 0 of culture) and replaced with serum-free growth medium as described before, except using DME containing 1000 mg of glucose/l (Sigma Chemical Company, St. Louis, MO). On day 4 of culture, medium was discarded, plates were rinsed twice with serum-free medium and cells were cultured in 1 ml fresh serum-free medium containing one of the following treatments: 10−10 , 10−8 or 10−6 M [Ala15 ]-h growth hormone-releasing factor-(1–29)NH2 (GRF; Sigma) or 10−9 , 10−8 or 10−7 M GnRH (Sigma) or 10−9 , 10−8 , 10−7 or 10−6 M NPY (porcine NPY; Sigma). In addition, cells were treated with the following combinations of 10−9 or 10−8 M GnRH with 10−8 , 10−7 or 10−6 M NPY or 10−8
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or 10−6 M GRF with 10−8 , 10−7 or 10−6 M NPY. Cells were exposed to treatment for 4 h at which time medium was harvested and quantified for LH and GH. 2.1. Radioimmunoassays Media samples were assayed in duplicate for GH [16] and LH [17] as previously described. Sensitivity of the assays was 0.4 and 0.15 ng/ml for GH and LH, respectively. Intraand interassay coefficients of variation were 4.5 and 10.2% for LH and 3.5 and 13% for GH, respectively. 2.2. Statistical analysis Basal secretion (control; C) was the amount of hormone secreted into the culture medium per 105 cells seeded/well in the absence of treatment or a secretagogue. Cells from each pig were cultured separately. Data were converted to percentage of basal secretion before averaging to minimize differences between wells. To obtain an estimate of variation between control wells, medium LH and GH concentrations for control wells were converted to a percentage of mean basal LH and GH concentration. This was then used to calculate a SE for basal secretion. Converted data were tested for homogeneity of variance using Hartley’s Fmax test [18]. Data were then subjected to a one way ANOVA and differences between means were determined by least-squares contrast [19].
3. Results Basal LH secretion (control; n = 7 pituitaries) was 12 ± 6 ng/well. Relative to control at 4 h, 10−9 , 10−8 and 10−7 M GnRH increased (P < 0.01) LH secretion by 169, 176 and 197%, respectively. Neuropeptide-Y did not alter (P > 0.4) basal (Fig. 1) nor 10−8 M GnRHinduced (Fig. 2) increase in LH secretion when compared to control or GnRH alone, respectively, but 10−9 M GnRH-stimulated LH secretion was reduced by NPY and was not different from control or GnRH alone (Fig. 2). Basal GH secretion (control; n = 7 pituitaries) was 56 ± 12 ng/well. Relative to control at 4 h, 10−10 , 10−8 and 10−6 M GRF increased GH secretion by 111%, 125% (P < 0.01) and 150% (P < 0.01), respectively. Only 10−6 M (134%) and 10−7 M (125%) NPY increased (P < 0.04) basal GH secretion (Fig. 3). Addition of 10−9 , 10−8 and 10−7 M NPY in combination with 10−8 M GRF suppressed (P < 0.04) GRF-stimulated GH secretion (Fig. 4). However, 10−9 M NPY enhanced (P < 0.06) the GH response to 10−6 M GRF (Fig. 4).
4. Discussion This is the first report to demonstrate a direct action of NPY on basal and GRF-induced GH secretion in anterior pituitary cells from prepuberal gilts. These results are in agreement with previous reports in which NPY increased GH secretion from perifused anterior
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Fig. 1. Effects of NPY on basal LH secretion. Values are the mean ± S.E. (n = 7 pituitaries). Control (C) = basal secretion in the absence of treatment.
pituitary cells in the rat [20], gold fish [21,22] and in static cultures derived from the rat at doses similar to those used in the present study. More importantly, NPY directly modulated pituitary response to GRF. In this regard, Peng et al. [22] demonstrated that pulses of NPY at 55 min intervals caused a desensitization of GH response in perifused goldfish
Fig. 2. Interaction of NPY with GnRH on LH secretion. Values are the mean ± S.E. (n = 7 pituitaries). Control (C) = basal secretion in the absence of treatment. Bars with different superscripts differ (P < 0.01).
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Fig. 3. Effects of NPY on basal GH secretion. Values are the mean ± S.E. (n = 7 pituitaries). Control (C) = basal secretion in the absence of treatment. a Different from C (P < 0.04).
pituitaries. In addition, SRIF treatment blocked the NPY induced increase in GH secretion demonstrating specificity of the GH response to NPY [22]. Although, static cultures were employed in the present study, the results reported support the idea that NPY modulates pituitary responsiveness to GRF.
Fig. 4. Interaction of NPY with GRF on GH secretion. Values are the mean ± S.E. (n = 7 pituitaries). Control (C) = basal secretion in the absence of treatment. a Different from C (P < 0.01). b Different from GRF alone (P < 0.06). c Different from GRF alone (P < 0.04).
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The apparent paradox in the action of NPY on basal and GRF-induced GH secretion may in part be explained by the presence of different intracellular signaling pathways and somatotrope cell populations. It has been reported that NPY acts to modulate extracellular Ca2+ influx in the rat gonadotrope [23] and suprachiasmatic neurons [24]. In the pig, two somatotrope subpopulations have been identified, a low density and high density population [25]. The low density somatotropes response to GRF is dependent on mobilization of both extra- and intracellular Ca2+ whereas, the high density somatotropes are primarily dependent on extracellular Ca2+ influx [26]. A recent report demonstrated that somatostatin reduced GRF-stimulated GH secretion but not basal secretion in low density somatotropes whereas, somatostatin did not alter GRF-induced GH release but stimulated basal GH secretion from high density somatotropes via an adenlyate cyclase/cAMP dependent pathway that was Ca2+ independent [27]. Somatostatin is not merely an inhibitor of GH secretion but may play a dual role in regulating GH secretion. Thus, it is conceivable that NPY may play a similar role in modulating GH secretion in the pig. The exact mechanism by which NPY exerts its effect on GH secretion is unclear. It is plausible that changes in pituitary sensitivity to GRF, in part, are the result of changing NPY concentration in portal blood or pituitary synthesis of NPY [28] in response to energy balance. Immunoreactive NPY cell bodies and fibers are located within the arcuate nuclei and ventromedial hypothalamus and fibers project to the median eminence in the pig [11,12]. Moreover, immunoreactive NPY has been detected in the anterior pituitary [12]. McDonald et al. [13] reported that the concentration of NPY in the hypophyseal portal blood of rats were three times greater than in systemic plasma. Fasting increased NPY concentration in the hypothalamus [29] and NPY mRNA levels in the arcuate nucleus [30,31]. Therefore, it is conceivable that NPY modulates temporal changes in GH secretion at the level of the pituitary that are observed during changes in energy balance. In the present study, NPY failed to influence either basal or 10−8 M GnRH-induced LH secretion, while the effect on the LH response to 10−9 M GnRH was equivocal. Similar results have been reported for the steer [32], ovariectomized, follicular and luteal phase heifers [33] and the anestrous ewe in which NPY failed to alter LH secretion directly or in combination with GnRH or estradiol from anterior pituitary cells in vitro [34]. Thus, results from the present study suggest that NPY does not modulate LH secretion from the anterior pituitary in the pig. In contrast, most reports in the rat on the effects of NPY are consistent with idea that NPY enhances basal LH secretion and the LH secretory response to GnRH [35–38] while this response varies with steroid environment [38,39]. In conclusion, under the experimental conditions employed in the present study, the results suggest that NPY directly stimulates basal GH secretion and modulates anterior pituitary responsiveness to GRF in the pig. However, there is no apparent effect of NPY on LH secretion from the pig anterior pituitary.
Acknowledgements The authors wish to thank Dr. A.F. Parlow, Harbor-UCLA medical Center, Torrance, CA for providing the porcine GH antiserum AFP-1021854. This research was supported by USDA funds. Mention of a trade name, proprietary product, or specific equipment does
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not constitute a guarantee or warranty by the US Department of Agriculture does not imply its approval to the exclusion of other products which may be suitable.
References [1] Billington CJ, Levine AS. Hypothalamic neuropeptide Y regulation of feeding and energy metabolism. Curr Opin Neurobiol 1992;2:847–51. [2] Clark JT, Kalra PS, Crowley WR, Kalra SP. Neuropeptide Y and human pancreatic polypeptide stimulated feeding behavior in rats. Endocrinology 1984;115:427–9. [3] Blum WF. Leptin: the voice of the adipose tissue. Horm Res 1997;48:2–8. [4] Jang M, Mistry A, Swick AG, Romsos DR. Leptin rapidly inhibits hypothalamic neuropeptide Y secretion and stimulates corticotropin-releasing hormone secretion in adrenalectomized mice. J Nutr 2000;130:2813–20. [5] Swart I, Overton JM, Houpt TA. The effect of food deprivation and experimental diabetes on orexin and NPY mRNA levels. Peptides 2001;22:2175–9. [6] Barb CR, Barrett JB, Kraeling RR, Rampacek GB. Role of leptin in modulating neuroendocrine function: a metabolic link between the brain–pituitary and adipose tissue. Reprod Domest Anim 1999;34:111–25. [7] Barb CR, Hausman GJ, Houseknecht KL. Biology of leptin in the pig. Domest Anim Endocrinol 2001;21:297–317. [8] Prunier A, Quesnel H. Nutritional influences on the hormonal control of reproduction in female pigs. Livest Prod Sci 2000;63:1–16. [9] Barb CR, Kraeling RR, Rampacek GB, Dove CR. Metabolic changes during the transition from the fed to the acute feed-deprived state in prepuberal and mature gilts. J Anim Sci 1997;75:781–9. [10] Barb CR, Barrett JB, Kraeling RR, Rampacek GB. Serum leptin concentrations, luteinizing hormone and growth hormone secretion during feed and metabolic fuel restriction in the prepuberal gilt. Domest Anim Endocrinol 2001;20:47–63. [11] Pearson PL, Anderson LL, Jacobson CD. The prepbertal ontogeny of neuropeptide Y-like immunoreactivity in the male Meishan pig brain. Dev Brain Res 1996;91:41–69. [12] Busch-Sorensen M, Shiekh SP, Tortora O, Schwartz TW, Gammeltoft S. Regional distribution of neuropeptide Y and its receptor in the porcine central nervous system. J Neurochem 1989;52:1545–52. [13] McDonald JK, Koenig JI, Gibbs DM, Collins P, Noe BD. High concentrations of neuropeptide Y in pituitary portal blood of rats. Neuroendocrinology 1987;46:538–41. [14] Sutton SW, Toyama TT, Otto S, Plotsky PM. Evidence that neuropeptide Y (NPY) released into the hypophysial–portal circulation participates in priming gonadotropes to the effects of gonadotropin releasing hormone (GnRH). Endocrinology 1988;123:1208–10. [15] Barb CR, Kraeling RR, Rampacek GB. Glucose and free fatty acid modulation of growth hormone and luteinizing hormone secretion by cultured porcine pituitary cells. J Anim Sci 1995;73:1416–23. [16] Barb CR, Estienne MJ, Kraeling RR, Marple DN, Rampacek GB, Rahe CH, et al. Endocrine changes in sows exposed to elevated ambient temperature during lactation. Domest Anim Endocrinol 1991;8:117–27. [17] Kesner JS, Kraeling RR, Rampacek GB, Johnson B. Absence of an estradiol-induced surge of luteinizing hormone in pigs receiving unvarying pulsatile gonadotropin-releasing hormone stimulation. Endocrinology 1987;121(5):1862–9. [18] Gill JL. In: Gill JL, editor. Design and analysis of experiments in the animal and medical sciences. Ames: Iowa State University Press; 1978. [19] SAS. SAS user’s guide (release 6.03). Cary, NC: Statistical Analysis Systems Institute Inc., 1988. [20] McDonald JK, Lumpkin MD, Smason WK, McCann SM. Neuropeptide Y affects secretion of luteinizing hormone and growth hormone in ovariectomized rats. Proc Natl Acad Sci 1985;82:561–4. [21] Peng C, Huang YP, Peter RE. Neuropeptide Y stimulates growth hormone and gonadotropin release from the goldfish pituitary in vitro. Neuroendocrinology 1990;52:28–34. [22] Peng C, Chang JP, Yu KL, Wong AOL, Van Goor F, Peter RE, et al. Neuropeptide-Y stimulates growth hormone and gonadotropin-II secretion in the goldfish pituitary: involvement of both presynaptic and pituitary cell actions. Endocrinology 1993;132:1820–9.
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[23] Shangold GA, Miller RJ. Direct neuropeptide Y-induced modulation of gonadotrope intracellular calcium transients and gonadotropin secretion. Endocrinology 1990;126:2336–42. [24] Obrietan K, Van den Pol AN. Neuropeptide Y depresses GABA-mediated calcium transients in developing suprachiasmatic nucleus neurons: a novel form of calcium long-term depression. J Neurosci 1996;16:3521–33. [25] Ramirez JL, Castano JP, Torronteras R, Martinez-Fuentes AJ, Frawley LS, Garcia-Navarro S, et al. Growth hormone (GH)-releasing factor differentially activates cyclic adenosine 3 ,5 -monophosphate- and inositol phosphate-dependent pathways to stimulate GH release in two porcine somatotrope subpopulations. Endocrinology 1999;140:1752–9. [26] Ramirez JL, Torronteras R, Malagon MM, Castano JP, Garcia-Navarro S, Gonzalez de Aguilar JL, et al. Growth hormone-releasing factor mobilizes cytosolic free calcium through different mechanisms in two somatotrope subpopulations from porcine pituitary. Cell Calcium 1988;23:207–17. [27] Ramirez JL, Gracia-Navarro F, Garcia-Navarro S, Torronteras R, Malagon MM, Castano JP. Somatostatin stimulates GH secretion in two porcine somatotrope subpopulations through a cAMP-dependent pathway. Endocrinology 2002;143:889–97. [28] Schwartz J. Intracellular communication in the anterior pituitary. Endocr Rev 2000;21:488–513. [29] Sahu A, Kalra PS, Kalra SP. Food deprivation and ingestion induce reciprocal changes in neuropeptide Y concentrations in the paraventricular nucleus. Peptides 1988;9:83–6. [30] Brady LS, Smith MA, Gold PW, Herkenham M. Altered expression of hypothalamic neuropeptide mRNAs in food-restricted and food-deprived rats. Neuroendocrinology 1990;52:441–7. [31] McShane TM, Petersen SL, McCrone S, Keisler DH. Influence of food restriction on neuropeptide-Y, proopiomelanocortin, and luteinizing hormone-releasing hormone gene expression in sheep hypothalami. Biol Reprod 1993;49:831–9. [32] Chao CC, Scribner KA, Dixon JE, Malven PV. Failure of neuropeptide Y to modulate the release of LH and prolactin by cultured bovine pituitary cells. Domest Anim Endocrinol 1987;4(4):309–14. [33] Denniston DJ, Thomas MG, Kane KK, Roybal CN, Canales L, Hallford DM, et al. Effect of neuropeptide Y on GnRH-induced LH release from bovine anterior pituitary cell cultures derived from heifers in a follicular, luteal or ovariectomized state. Anim Reprod Sci 2003;78:25–31. [34] Brooks AN, Graham BJM, Naylor AM. Interactions between neuropeptide Y, luteinizing hormone-releasing hormone and estradiol in the control of luteinizing hormone release from cultured ovine pituitary cells. Peptides 1991;12:397–400. [35] Bauer-Dantoin AC, Knox KL, Schwartz NB, Levine JE. Estrous cycle stage-dependent effects of neuropeptide-Y on luteinizing hormone (LH)-releasing hormone-stimulated LH and follicle-stimulating hormone secretion from anterior pituitary fragments in vitro. Endocrinology 1993;133:2413–7. [36] Leiva LA, Croxatto HB. Comparison of the effect of hypothalamic neuropeptides upon luteinizing hormone secretion by cultured rat anterior pituitary cells. Biol Res 1994;27:113–21. [37] Knox KL, Bauer-Dantoin AC, Levine JE, Schwartz NB. Unmasking of neuropeptide-Y inhibitory effects on in vitro gonadotropin secretion from pituitaries of metestrous, but not proestrous, rats. Endocrinology 1995;136:87–194. [38] Leupen SM, Levine JE. Role of protein kinase C in facilitation of luteinizing hormone (LH)-releasing hormone-induced LH surges by neuropeptide Y. Endocrinology 1999;140:3682–7. [39] Hill JW, Urban JH, Xu M, Levine JE. Estrogen induces neuropeptide Y (NPY) Y1 receptor gene expression and responsiveness to NPY in gonadotrope-enriched pituitary cell cultures. Endocrinology 2004;145:2283–90.
Neuropeptide Y modulates growth hormone but not luteinizing hormone secretion from prepuberal gilt anterior pituitary cells in culture C.R. Barb ∗ , J.B. Barrett 1 USDA-ARS, Animal Physiology Research Unit, Russell Agricultural Research Center, P.O. Box 5677, Athens, GA 30604-5677, USA Received 1 December 2004; received in revised form 1 March 2005; accepted 16 March 2005
Abstract Pituitary cells, from seven 160- to 170-day-old pigs, were studied in primary culture to determine the affects NPY on LH and GH secretion at the level of the pituitary. On day 4 of culture, medium was discarded, plates were rinsed twice with serum-free medium and cells were cultured in 1 ml fresh medium without serum and challenged individually with 10−10 , 10−8 or 10−6 M [Ala15 ]-h growth hormone-releasing factor-(1–29)NH2 (GRF); 10−9 , 10−8 or 10−7 M GnRH or 10−9 , 10−8 , 10−7 or 10−6 M NPY individually or in combinations with 10−9 or 10−8 M GnRH or 10−8 or 10−6 M GRF. Cells were exposed to treatment for 4 h at which time medium was harvested and quantified for LH and GH. Basal LH secretion (control; n = 7 pituitaries) was 12 ± 6 ng/well. Relative to control at 4 h, 10−9 , 10−8 and 10−7 M GnRH increased (P < 0.01) LH secretion by 169, 176 and 197%, respectively. Neuropeptide-Y did not alter (P > 0.4) basal LH secretion nor 10−8 M GnRH-induced increase in LH secretion but 10−9 M GnRH-stimulated LH secretion was reduced by NPY and was not different from control or GnRH alone. Basal GH secretion (control; n = 7 pituitaries) was 56 ± 12 ng/well. Relative to control at 4 h, 10−10 , 10−8 and 10−6 M GRF increased GH secretion by 111%, 125% (P < 0.01) and 150% (P < 0.01), respectively. Only 10−6 M (134%) and 10−7 M (125%) NPY increased (P < 0.04) basal GH secretion. Addition of 10−9 , 10−8 and 10−7 M NPY in combination with 10−8 M GRF suppressed (P < 0.04) GRF-stimulated GH secretion. However, 10−9 M NPY enhanced (P < 0.06) the ∗
Corresponding author. Tel.: +1 706 583 8276. E-mail address: [email protected] (C.R. Barb). 1 Present address: USDA-ARS, Antimicrobial Resistance Research Unit, Russell Research Center, P.O. Box 5677, Athens, GA 30604-5677, USA. 0739-7240/$ – see front matter. Published by Elsevier Inc. doi:10.1016/j.domaniend.2005.03.004
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GH response to 10−6 M GRF. These results demonstrate that NPY may directly modulate GH secretion at the level of the pituitary gland. Published by Elsevier Inc. Keywords: Neuropeptide Y; Pituitary; Luteinizing hormone; Growth hormone; Pig
1. Introduction Neuropeptide-Y (NPY) is a potent regulator of feeding, energy expenditure and fat storage [1,2]. Furthermore, hypothalamic NPY is responsive to changes in energy balance and blood leptin concentrations [3–5]. Intracerebroventricular (ICV) administration of NPY suppressed luteinizing hormone (LH) secretion and increased growth hormone (GH) secretion and feed intake in the prepuberal gilt [6,7]. Moreover, in the prepuberal gilt feed restriction delayed the onset of puberty [8], increased GH levels [9] and suppressed leptin secretion [10]. Immunoreactive NPY cell bodies and fibers are located within the arcuate nuclei, ventromedial hypothalamus and median eminence [11]. The presence of NPY in the anterior pituitary in the pig [12] would suggest that hypothalamic NPY is transported via the hypophyseal portal blood circulation to the anterior pituitary. In support of this idea, NPY has been detected in the hypophyseal portal blood in the rat [13,14]. The above reports are consistent with a direct effect of NPY on pituitary hormone secretion. Thus, NPY may relay metabolic information to the brain–pituitary axis to regulate pituitary hormone secretion. Therefore, the present study was conducted to test the hypothesis that NPY acts directly on the anterior pituitary to modulate LH and GH secretion in the prepuberal gilt. 2. Materials and methods Pituitary glands were aseptically removed from seven intact prepuberal gilts 160 to 170 days of age. Ovaries were examined at slaughter and gilts were considered prepuberal because their ovaries were devoid of corpora albicantia and corpora lutea. All subsequent procedures were performed under sterile conditions. The anterior lobe was dissected from each pituitary gland; cells enzymatically dispersed and cultured as previously described [15]. Cells from each pig were cultured separately. Cell viability and number were assessed by counting cells which excluded trypan blue on a hemocytometer. Cell viability was 96%. Cells were diluted and plated, based on the number of live cells. Cells were plated 105 cells/ml with culture medium in a 24-well cluster plate. Culture medium was changed on day 3 (day of seeding = day 0 of culture) and replaced with serum-free growth medium as described before, except using DME containing 1000 mg of glucose/l (Sigma Chemical Company, St. Louis, MO). On day 4 of culture, medium was discarded, plates were rinsed twice with serum-free medium and cells were cultured in 1 ml fresh serum-free medium containing one of the following treatments: 10−10 , 10−8 or 10−6 M [Ala15 ]-h growth hormone-releasing factor-(1–29)NH2 (GRF; Sigma) or 10−9 , 10−8 or 10−7 M GnRH (Sigma) or 10−9 , 10−8 , 10−7 or 10−6 M NPY (porcine NPY; Sigma). In addition, cells were treated with the following combinations of 10−9 or 10−8 M GnRH with 10−8 , 10−7 or 10−6 M NPY or 10−8
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or 10−6 M GRF with 10−8 , 10−7 or 10−6 M NPY. Cells were exposed to treatment for 4 h at which time medium was harvested and quantified for LH and GH. 2.1. Radioimmunoassays Media samples were assayed in duplicate for GH [16] and LH [17] as previously described. Sensitivity of the assays was 0.4 and 0.15 ng/ml for GH and LH, respectively. Intraand interassay coefficients of variation were 4.5 and 10.2% for LH and 3.5 and 13% for GH, respectively. 2.2. Statistical analysis Basal secretion (control; C) was the amount of hormone secreted into the culture medium per 105 cells seeded/well in the absence of treatment or a secretagogue. Cells from each pig were cultured separately. Data were converted to percentage of basal secretion before averaging to minimize differences between wells. To obtain an estimate of variation between control wells, medium LH and GH concentrations for control wells were converted to a percentage of mean basal LH and GH concentration. This was then used to calculate a SE for basal secretion. Converted data were tested for homogeneity of variance using Hartley’s Fmax test [18]. Data were then subjected to a one way ANOVA and differences between means were determined by least-squares contrast [19].
3. Results Basal LH secretion (control; n = 7 pituitaries) was 12 ± 6 ng/well. Relative to control at 4 h, 10−9 , 10−8 and 10−7 M GnRH increased (P < 0.01) LH secretion by 169, 176 and 197%, respectively. Neuropeptide-Y did not alter (P > 0.4) basal (Fig. 1) nor 10−8 M GnRHinduced (Fig. 2) increase in LH secretion when compared to control or GnRH alone, respectively, but 10−9 M GnRH-stimulated LH secretion was reduced by NPY and was not different from control or GnRH alone (Fig. 2). Basal GH secretion (control; n = 7 pituitaries) was 56 ± 12 ng/well. Relative to control at 4 h, 10−10 , 10−8 and 10−6 M GRF increased GH secretion by 111%, 125% (P < 0.01) and 150% (P < 0.01), respectively. Only 10−6 M (134%) and 10−7 M (125%) NPY increased (P < 0.04) basal GH secretion (Fig. 3). Addition of 10−9 , 10−8 and 10−7 M NPY in combination with 10−8 M GRF suppressed (P < 0.04) GRF-stimulated GH secretion (Fig. 4). However, 10−9 M NPY enhanced (P < 0.06) the GH response to 10−6 M GRF (Fig. 4).
4. Discussion This is the first report to demonstrate a direct action of NPY on basal and GRF-induced GH secretion in anterior pituitary cells from prepuberal gilts. These results are in agreement with previous reports in which NPY increased GH secretion from perifused anterior
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Fig. 1. Effects of NPY on basal LH secretion. Values are the mean ± S.E. (n = 7 pituitaries). Control (C) = basal secretion in the absence of treatment.
pituitary cells in the rat [20], gold fish [21,22] and in static cultures derived from the rat at doses similar to those used in the present study. More importantly, NPY directly modulated pituitary response to GRF. In this regard, Peng et al. [22] demonstrated that pulses of NPY at 55 min intervals caused a desensitization of GH response in perifused goldfish
Fig. 2. Interaction of NPY with GnRH on LH secretion. Values are the mean ± S.E. (n = 7 pituitaries). Control (C) = basal secretion in the absence of treatment. Bars with different superscripts differ (P < 0.01).
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Fig. 3. Effects of NPY on basal GH secretion. Values are the mean ± S.E. (n = 7 pituitaries). Control (C) = basal secretion in the absence of treatment. a Different from C (P < 0.04).
pituitaries. In addition, SRIF treatment blocked the NPY induced increase in GH secretion demonstrating specificity of the GH response to NPY [22]. Although, static cultures were employed in the present study, the results reported support the idea that NPY modulates pituitary responsiveness to GRF.
Fig. 4. Interaction of NPY with GRF on GH secretion. Values are the mean ± S.E. (n = 7 pituitaries). Control (C) = basal secretion in the absence of treatment. a Different from C (P < 0.01). b Different from GRF alone (P < 0.06). c Different from GRF alone (P < 0.04).
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The apparent paradox in the action of NPY on basal and GRF-induced GH secretion may in part be explained by the presence of different intracellular signaling pathways and somatotrope cell populations. It has been reported that NPY acts to modulate extracellular Ca2+ influx in the rat gonadotrope [23] and suprachiasmatic neurons [24]. In the pig, two somatotrope subpopulations have been identified, a low density and high density population [25]. The low density somatotropes response to GRF is dependent on mobilization of both extra- and intracellular Ca2+ whereas, the high density somatotropes are primarily dependent on extracellular Ca2+ influx [26]. A recent report demonstrated that somatostatin reduced GRF-stimulated GH secretion but not basal secretion in low density somatotropes whereas, somatostatin did not alter GRF-induced GH release but stimulated basal GH secretion from high density somatotropes via an adenlyate cyclase/cAMP dependent pathway that was Ca2+ independent [27]. Somatostatin is not merely an inhibitor of GH secretion but may play a dual role in regulating GH secretion. Thus, it is conceivable that NPY may play a similar role in modulating GH secretion in the pig. The exact mechanism by which NPY exerts its effect on GH secretion is unclear. It is plausible that changes in pituitary sensitivity to GRF, in part, are the result of changing NPY concentration in portal blood or pituitary synthesis of NPY [28] in response to energy balance. Immunoreactive NPY cell bodies and fibers are located within the arcuate nuclei and ventromedial hypothalamus and fibers project to the median eminence in the pig [11,12]. Moreover, immunoreactive NPY has been detected in the anterior pituitary [12]. McDonald et al. [13] reported that the concentration of NPY in the hypophyseal portal blood of rats were three times greater than in systemic plasma. Fasting increased NPY concentration in the hypothalamus [29] and NPY mRNA levels in the arcuate nucleus [30,31]. Therefore, it is conceivable that NPY modulates temporal changes in GH secretion at the level of the pituitary that are observed during changes in energy balance. In the present study, NPY failed to influence either basal or 10−8 M GnRH-induced LH secretion, while the effect on the LH response to 10−9 M GnRH was equivocal. Similar results have been reported for the steer [32], ovariectomized, follicular and luteal phase heifers [33] and the anestrous ewe in which NPY failed to alter LH secretion directly or in combination with GnRH or estradiol from anterior pituitary cells in vitro [34]. Thus, results from the present study suggest that NPY does not modulate LH secretion from the anterior pituitary in the pig. In contrast, most reports in the rat on the effects of NPY are consistent with idea that NPY enhances basal LH secretion and the LH secretory response to GnRH [35–38] while this response varies with steroid environment [38,39]. In conclusion, under the experimental conditions employed in the present study, the results suggest that NPY directly stimulates basal GH secretion and modulates anterior pituitary responsiveness to GRF in the pig. However, there is no apparent effect of NPY on LH secretion from the pig anterior pituitary.
Acknowledgements The authors wish to thank Dr. A.F. Parlow, Harbor-UCLA medical Center, Torrance, CA for providing the porcine GH antiserum AFP-1021854. This research was supported by USDA funds. Mention of a trade name, proprietary product, or specific equipment does
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not constitute a guarantee or warranty by the US Department of Agriculture does not imply its approval to the exclusion of other products which may be suitable.
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