373 Interaction between substance P and TRH in the control of prolactin release B H Duvilanski, D Pisera, A Seilicovich, M del Carmen Díaz, M Lasaga, E Isovich and M O Velardez Centro de Investigaciones en Reproducción, Facultad de Medicina, Universidad de Buenos Aires, Argentina (Requests for offprints should be addressed to B H Duvilanski, Centro de Investigaciones en Reproducción, Facultad de Medicina, Piso 10, Universidad de Buenos Aires, Paraguay 2155 (1121) Buenos Aires, Argentina; Email: [email protected]) Abstract Substance P (SP) may participate as a paracrine and/or release. In order to study the interaction between TRH autocrine factor in the regulation of anterior pituitary and SP on prolactin release, anterior pituitaries were function. This project studied the effect of TRH on SP incubated with either TRH (107 M) or SP, or with both content and release from anterior pituitary and the role of peptides. SP (107 and 106 M) by itself stimulated SP in TRH-induced prolactin release. TRH (107 M), prolactin release. While 107 M SP did not modify the but not vasoactive intestinal polypeptide (VIP), increased TRH effect, 106 M SP reduced TRH-stimulated pro- immunoreactive-SP (ir-SP) content and release from male lactin release. SP (105 M) alone failed to stimulate rat anterior pituitary in vitro. An anti-prolactin serum also prolactin release and markedly decreased TRH-induced increased ir-SP release and content. In order to determine prolactin release. The present study shows that TRH whether intrapituitary SP participates in TRH-induced stimulates ir-SP release and increases ir-SP content in the prolactin release, anterior pituitaries were incubated with anterior pituitary. Our data also suggest that SP may act as TRH (107 M) and either WIN 62,577, a specific a modulator of TRH effect on prolactin secretion by a antagonist of the NK1 receptor, or a specific anti-SP paracrine mechanism. 8 7 serum. Both WIN 62,577 (10 and 10 M) and the Journal of Endocrinology (2000) 166, 373–380 anti-SP serum (1:250) blocked TRH-induced prolactin Introduction rats (Yoshikawa & Hong 1983, Jessop et al. 1992). SP content in the anterior pituitary is regulated by several Substance P (SP), a neuropeptide belonging to the tachy- circulating hormones (De Palatis et al. 1985, Jonnassen kinin family, is found in most tissues, and produces a broad et al. 1987, Aronin et al. 1988, O’Halloran et al. 1990, range of cellular responses. SP exerts its effects through Jones et al. 1994). a specific receptor in a G protein-linked family of SP modulates several anterior pituitary hormones (Jessop receptors. It enhances polyphosphoinositide breakdown et al. 1992). SP stimulates in vitro prolactin release from the and increases intracellular free calcium levels (Mau et al. anterior pituitary (Vijayan & McCann 1979, Seilicovich 1990, Mau & Saermark 1991). In the anterior pituitary, et al. 1993), while the blockade of intrapituitary SP with the preferred SP receptor, a neurokinin-1 (NK1) subtype specific SP antibodies decreases basal prolactin release of the tachykinin receptor family, is found exclusively in (Afione et al. 1990a). lactotrophs and gonadotrophs (Larsen et al. 1992). Thyrotropin-releasing hormone (TRH) is a hypo- SP is synthesized (Jessop et al. 1992) and released physiotropic neuropeptide that increases thyrotropin (Seilicovich et al. 1990, Arita et al. 1993) by the anterior (TSH) and prolactin secretion. TRH is synthesized not pituitary. SP was found to be localized only in only in the hypothalamus but also in a subpopulation of somatotrophs and thyrotrophs of male rats (Brown et al. somatotrophs in the anterior pituitary (Bruhn et al. 1994). 1991, Arita et al. 1994). Also SP-like immunoreactive Several studies have demonstrated the modulatory role innervation is present in the anterior pituitary, distributed of some pituitary peptides on hypothalamic neurotransmit- around secretory cells such as lactotrophs (Liu et al. 1996) ters and neuropeptides that regulate anterior pituitary and corticotrophs (Ju et al. 1991) and around blood vessels hormone secretion (Peillon et al. 1997). Interactions (Mikkelsen et al. 1989). between neurotransmitters and SP or vasoactive intestinal SP content and preprotachykinin mRNA vary with age polypeptide (VIP) at pituitary level (Afione et al. 1990a,b, and sex of the animals; it is higher in male than in female Duvilanski et al. 1990, Apfelbaum 1998), and also Journal of Endocrinology (2000) 166, 373–380 Online version via http://www.endocrinology.org 0022–0795/00/0166–373 2000 Society for Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 09/24/2021 11:21:02AM via free access 374 B H DUVILANSKI and others · Interaction between SP and TRH between VIP and TRH (Balsa et al. 1996) have been dry ice and processed for SP determination. An aliquot of described. each medium was separated before acidification and stored Since SP has a paracrine effect on prolactin secretion, at 20 C until prolactin determination. the aim of this study was to ascertain whether anterior pituitary SP is involved in the effect of TRH on prolactin release. Therefore, we designed our experiments in order Static incubation of anterior pituitary glands to study: (1) the influence of SP blockade on the effect of Three pituitaries per tube were used to assay SP and TRH on prolactin release; (2) the effect of TRH on prolactin release in each incubation, and only one when anterior pituitary SP release and content; and (3) the prolactin release alone was studied. The pituitaries were interaction between TRH and SP in the control of pre-incubated for 60 min in 1 ml KRB buffer. Afterwards, prolactin release. the tissue was incubated for another 60 min, unless other- wise indicated, in 1 ml of fresh KRB buffer containing the different drugs studied. Controls were incubated with Materials and Methods medium alone or vehicle or normal rabbit serum (NRS). At the end of the incubation period, media were aspirated. Male Wistar rats (200–250 g) were kept in controlled Media and tissues were immediately acidified, frozen on conditions of light (12 h light : 12 h darkness) and tem- dry ice and processed for SP determination. An aliquot of perature (21–24 C). Lab chow and water were supplied medium was separated before acidification, and stored at and animals were allowed to feed ad libitum. The rats were 20 C until prolactin determination. WIN 62,577 is a kept according to the NIH Guide for the Care and Use of non-peptide NK1 receptor antagonist that binds to rat Laboratory Animals. Rats were killed by decapitation and brain homogenates with K values of 21 nM (Appel et al. the anterior pituitary lobes removed, then placed in i 1991). WIN 62,577 was dissolved in DMSO and diluted Krebs–Ringer bicarbonate buffer, pH 7·4, containing in KRB buffer. A final concentration of 0·02% DMSO 10 mM glucose, 25 mM Hepes, 0·1 mM bacitracin, 0·1% had no effect on basal prolactin release. ascorbic acid and 0·1% bovine serum albumin (KRB buffer). All drugs were purchased from Sigma (St Louis, MO, SP determination USA), except TRH, SP and VIP (Peninsula Lab. Inc., Belmont, CA, USA), and WIN 62,577 (Research Media and tissues were heated at 100 C for 5 min in 1 M Biochemical Int., Natick, MA, USA). Rat prolactin acetic acid. The tissues were then homogenized and both and NIDDK anti-prolactin serum (S-9) were kindly media and tissues centrifuged at 14 500 r.p.m. for 20 min. supplied by the National Hormone and Pituitary Program The supernatants were lyophilized and stored at 70 C (Torrance, CA, USA). until SP determination. SP was measured by double Rabbit anti-SP serum was prepared, characterized antibody radioimmunoassay with 125I-SP as tracer, as and tested as previously reported (Debeljuk et al. 1988). previously described (Seilicovich et al. 1990). The intra- This anti-serum showed no cross-reactivity with other and interassay coefficients of variation were under 10%. mammalian neuropeptides such as neurokinin A, neuro- Immunoreactive SP (ir-SP) was expressed as ng/mg pro- kinin B, VIP, neuromedin B and neurotensin when these tein. Protein concentration of tissue pellets was determined peptides were tested at doses 5000–10 000-fold higher by Lowry’s method. than those of SP. Prolactin determination Dynamic incubation of anterior pituitary glands. Pulse-chase experiments Prolactin was measured in the incubation medium by a double antibody radioimmunoassay, with kits provided by Five pituitaries were placed into a small perforated basket the National Hormone and Pituitary Program. Results and immersed in a tube containing 1 ml of KRB buffer were expressed in terms of rat prolactin RP-3 standard. and pre-incubated for 30 min at 37 C, in an atmosphere The intra- and interassay coefficients of variation were less of 95% O –5% CO , with constant shaking of 60 cycles 2 2 than 9%. per min. Basal release was then determined by incubating the baskets for two periods of 15 min in fresh KRB buffer. Afterwards, the baskets were transferred to a tube contain- ing TRH (107 M) and incubated for 5 min (TRH Statistical analysis pulse). Finally, post-stimulus release was assessed by incu- Results were expressed as means...s and evaluated bating the baskets for two periods of 15 min in KRB by the Student t-test or by one- or two-way analysis of buffer. Controls were run in parallel with KRB buffer variance (ANOVA) followed by Student–Newman–Keuls only. The media were immediately acidified, frozen on multiple comparison test for unequal replicates or Journal of Endocrinology (2000) 166, 373–380 www.endocrinology.org Downloaded from Bioscientifica.com at 09/24/2021 11:21:02AM via free access Interaction between SP and TRH · B H DUVILANSKI and others 375 Table 1 Effect of WIN 62,577 on basal and SP-induced prolactin release.