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Cell immunoblot assay study demonstrating the release of PACAP from individual anterior pituitary cells of rats and the effect of PACAP on LH release
Regulatory Peptides 109 (2002) 75 – 81 www.elsevier.com/locate/regpep
Cell immunoblot assay study demonstrating the release of PACAP from individual anterior pituitary cells of rats and the effect of PACAP on LH release ´ . Nemeske´ri a, A. Heinzlmann a, N. Suzuki b, A. Arimura c, K. Ko¨ves a,* E. Szabo´ a, A a
Department of Human Morphology and Developmental Biology, Faculty of Medicine, Semmelweis University, Tuzolto u.58, H-1094 Budapest, Hungary b Discovery Research Laboratories, Takeda Chemical Industries, Ltd., Tsukuba, Japan c US-Japan Biomedical Research Laboratories, Tulane University Hebert Center, Belle Chasse and Department of Physiology, Tulane University Medical School, New Orleans, LA, USA
Abstract The presence of pituitary adenylate cyclase activating polypeptide (PACAP) was previously demonstrated in the anterior pituitary by radioimmunoassay, immunohistochemistry, and reverse transcript-polymerase chain reaction (RT-PCR). With the use of cell immunoblot assay (CIBA), when the pituitary cells were cultured on nitrocellulose membrane, the release of PACAP by individual anterior pituitary cells was observed. The released peptide, trapped by the nitrocellulose membrane forming a blot around the cells, was demonstrated by immunocytochemistry. Double labeling revealed that a part of PACAP-immunoreactive cells can release LH as well. With the use of sandwich enzyme immunoassay (S-EIA), it was found that the concentration of PACAP in the anterior pituitaries is 10 10 M. In cell culture in a similar concentration, PACAP stimulated the LH release from female gonadotropes, but did not influence it from male ones. The stimulated release of LH was indicated by the enhancement in the diameter of LH blots compared to the untreated control cultures. We concluded that PACAP may be released from the anterior pituitary cells in a concentration which would be able to influence LH release not only in vitro but under in vivo conditions as well. The effect of PACAP on LH release was different in female and male pituitary cultures. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Immunocytochemistry; Cell immunoblot assay (CIBA); Sandwich enzyme immunoassay (S-EIA); Anterior pituitary; Rat
1. Introduction Pituitary adenylate cyclase activating polypeptide (PACAP) was isolated on the basis of its ability to stimulate adenylate cyclase in primary anterior pituitary cell cultures [1]. PACAP is present in two bioactive amidated forms, PACAP38 and PACAP27, with 38 and 27 amino acid residues, respectively [2]. It was also demonstrated that PACAP stimulates the release of several anterior pituitary hormones, among them LH, although the results published in various papers were not consistent. The models in which the effect of PACAP was studied are very different. In vivo the effect of PACAP on LH release depends on the form of PACAP (whether PACAP38 or PACAP27 *
Corresponding author. Tel.: +36-1-215-6920x3618; fax: +36-1-2153064. E-mail address: [email protected] (K. Ko¨ves).
was administered), on the species, on the dose and on the route of the administration. PACAP38 administered in an intravenous (i.v.) infusion to male rats, stimulates LH secretion [3]. When PACAP38 was given intraarterially to ovariectomized ewes, it had no effect; however, its intracerebroventricular (i.c.v.) administration inhibited LH pulse frequency and amplitude [4]. Before the critical period of the proestrous stage, i.v. administration of PACAP38 to female rats did not influence the ovulation; however, its i.c.v. administration inhibited the expected ovulation [5,6]. In the same model, i.c.v. administration of PACAP27 did not inhibit the ovulation, but enhanced the proestrous LH surge and the number of expelled ova [7]. In vitro in a static primary anterior pituitary cell culture, PACAP38 did not influence the LH release, although in a superfusion system there was a mild stimulation [1]. In perifused rat anterior pituitary cells deriving from orchid-
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ectomized male rats, PACAP38 enhanced the LH release when it was added to the medium in a pulsatile pattern [8]. In a gonadotrop cell line, PACAP38 potentiated the effect of LHRH on LH release [9]. The source of PACAP in living organism may be the hypothalamus and the anterior pituitary itself. Not in intact rats, but under special circumstances (hypophysectomy, pituitary stalk section, or the removal of the eyes), we have observed the presence of PACAP-immunoreactive fibers in the external zone of the median eminence (ME) [10 – 12]. It was also demonstrated that PACAP was released into the portal blood both in male and female rats [13]. In our laboratory, it was observed that PACAP immunoreactivity is also present in the anterior pituitary of male rats. Double labeling revealed that PACAP colocalized with LH and FSH immunoreactivities in pituitary sections. In female rats, PACAP-immunoreactive cells could only be stained when the animals were sacrificed on the day of proestrous [14]. These findings suggest that the pituitary-born PACAP, similarly to the hypothalamic one [5– 7], is also involved in the regulation of the gonadotrop hormone secretion. In the present experiment, we have studied the role of PACAP in LH secretion of male and proestrous female anterior pituitaries. The aim of our work was to investigate:
whether our method, the cell immunoblot assay, is suitable for demonstrating the PACAP release of individual anterior pituitary cells, whether PACAP-immunoreactive cells can release LH as well (it was examined by CIBA and double-labeling immunocytochemistry), what the physiological concentration of PACAP is in the anterior pituitary (it was measured with the use of sandwich enzyme immunoassay [S-EIA]), how and in what concentration PACAP influences LH release from the gonadotropes of male or female pituitaries and whether it is similar to the physiological one (it was examined by CIBA and analysed by the Image ProPlus program).
2. Materials and methods 2.1. Animals Adult (3– 4 months old) Sprague – Dawley (CRL: CD) male and proestrous female rats were used for the experiments. The animals were kept in a temperature- (22 F 2 jC) and light-controlled (lights on at 05:00 and lights off at 19:00) vivarium. The male rats were randomly chosen. Only those female rats were used for the experiments which showed regular ovarian cyclicity. Treating the animals was in accordance with the rules of the ‘‘European convention for the protection of vertebrate animals used
for experimental and other scientific purposes’’, Strasbourg, 1986. 2.2. Cell immunoblot assay (CIBA) 2.2.1. Cell culturing CIBA was carried out according to Arita et al. [15] and Cimini et al. [16] modified by one of us (Nemeske´ri). This method is suitable to demonstrate the hormone release of individual cells. Two male and two proestrous female rats were decapitated, the pituitaries were removed immediately under sterile conditions and dispersed in freshly prepared trypsin solution (Sigma-T4799) containing 0.11% trypsin in minimum essential medium (MEM) – Eagle (GIBCO EUROPE) and 0.1% bovine serum albumin (BSA). The dispersion was carried out in a Spinner flask (Bellco). We obtained cell yields of approximately 1.8 –2.4 106 cells/ pituitary of rats, with a cell viability of more than 90%. The viability of the cells was checked by vital staining. Pieces of 5 5 mm supported nitrocellulose membranes (BIO-RAD Laboratories) were placed on the surface of a metal grid in tissue culture dish (Greiner Labortechnik— 35 mm, sterile) containing 650 Al of Dulbecco-MEM (DMEM) (GIBCO BRL). Seven thousand monodispersed pituitary cells in a drop of 15 Al DMEM were placed onto the nitrocellulose membranes and cultured in 5% CO2 – 95% air incubator for 22 h. During the culturing period, the released hormones and peptides were trapped by the nitrocellulose membrane giving a possibility for immunostaining (see below). To a half of the wells containing cultured cells, PACAP was added in 10 7 M concentration. The concentration of PACAP in the media at the end of the culturing was measured by S-EIA in those wells to which PACAP was added (see later). 2.2.2. Immunocytochemistry (ICC) After the incubation period, the nitrocellulose membranes were fixed in buffered 4% paraformaldehyde solution. Those membranes that were cultured in DMEM without PACAP were immunostained for PACAP or LH. Those that were treated with PACAP were only stained for LH. The immunostaining was carried out according to the directions of the Vectastain ABC KIT (Vector Laboratories, Burlingame, CA). Antibody binding sites were visualized using H2O2 and diaminobenzidine-tetrahydrochloride (DAB). PACAP antiserum was raised in rabbit and previously characterized [14,17]. LH antiserum was raised in guinea pig and provided by NIDDK and NHPP (University of Maryland, School of Medicine, Baltimore, MD). The dilution of the PACAP antiserum was 1:6000, and that of the LH antiserum was 1:10 000. Selected membranes demonstrating PACAP blots were also stained for LH immunoreactivity using an indirect immunofluorescent method and FITC conjugate. In this latter case, the LH antiserum was used at 1:500 dilution.
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2.3. Image analysis In randomly chosen 10 viewfields of cell cultures deriving from two male and two female animals, the number and the diameter of PACAP and LH blots were analysed with the help of the Image ProPlus program (Media Cybernetics, USA). Statistical significance was calculated by the proposal of the Prizma Program. 2.4. Sandwich (two-site) enzyme immunoassay (S-EIA) 2.4.1. Preparation of the pituitary samples Fifteen male rats were sacrificed by decapitation. The pituitaries were removed immediately. The anterior pituitaries were separated, the intermediate and the posterior lobes were discarded. The anterior pituitaries were frozen on dry ice and stored at 70 jC. Before the S-EIA, the pituitaries were thawed. Three pituitaries were pooled and homogenized by ultrasound homogenizer (Ultrasonic Homogenizer, Chicago) in trifluoroacetic acid (TFA). The homogenates were centrifuged at 12 000 rpm for 20 min at 4 jC. The supernatants were dried using speed vac, then washed and dried one more time to remove TFA. The PACAP content of the samples was measured. 2.4.2. Cell culture media The PACAP content of those cell culture media to which PACAP was added was also measured at the end of the incubation period.
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2.4.3. S-EIA protocol The standard peptide (PACAP38) and the samples were put in the wells of a PA-6N (monoclonal antibody against N-terminal portion of PACAP38)-coated microtest ELISA plate and incubated for 96 h at 4 jC. The wells were washed in PBS (phosphate buffer, 0.02 M, pH 7, containing 1% bovine serum albumine). At the end of the incubation period, the wells were reacted with HRP-labeled PA-2C (monoclonal antibody against the C-terminal portion of PACAP38) for 24 h at 4 jC and were washed in PBS. The activity of HRP, bound on each well, was measured with the TMB microwell peroxidase system (Kirkegaad & Perry Labs, USA). The optical absorbance was read at 490 nm by an ELISA reader. The monoclonal antibodies against PACAP38 (PA-6N and PA-2C) were raised and tested in Suzuki’s laboratory [18].
3. Results 3.1. Release of PACAP from individual anterior pituitary cells In the cell cultures with the use of CIBA, PACAPimmunoreactive cells were observed, a few of which released PACAP. The immunoreactive PACAP in cultures were visualized by DAB chromogen. The PACAP-immunoreactive cells exhibited different releasing activity in both male and proestrous female cultures (Fig. 1). The released
Fig. 1. PACAP blots deriving from male (A and B) and proestrous female rats (C and D). In A, a PACAP-immunoreactive cell shows a sharp contour in the middle of a blot (indicated by arrow). In B, C and D, arrowheads indicate blots in which the cell contour cannot be recognized and arrows show PACAPimmunoreactive cells which did not release PACAP. 300 .
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of the double-labeled blots, a bright fluorescent halo indicated the release of LH. In the middle of these blots, the dark DAB reaction product with an irregular contour indicated the released PACAP. The amount of the released PACAP was moderate compared to the LH released by the same gonadotropes. One of them is seen in Fig. 2A. In other double-labeled blots in the middle of the bright fluorescent halo, the contour of PACAP immunoreactivity was sharp, which indicated that these gonadotropes did not release its PACAP content. One of them is seen in Fig. 2B. 3.3. PACAP content in the anterior pituitaries To study the physiological concentration of PACAP in the anterior pituitary, S-EIA was used. The level of PACAP in the anterior pituitaries was measurable. In the pooled pituitary samples, PACAP was present in 10 10 M concentration (Fig. 3). 3.4. Effect of PACAP on LH release by individual gonadotropes
Fig. 2. Double labeling for PACAP and LH in pituitary cell cultures. In A, both PACAP (dark irregular DAB reaction product, arrowhead) and LH (bright fluorescent halo, arrow) blots can be seen. This cell released a moderate amount of PACAP and a larger amount of LH. In B, the sharp contour of PACAP-immunoreactive cell (dark DAB reaction product, arrowhead) can be seen in the middle of the LH blot (bright fluorescent halo, arrow) indicating that PACAP was synthetised but not released by this cell. 900 .
PACAP formed blots around the cells. There were blots in which the contour of the cell was relatively sharp indicating that these cells did not completely release their PACAP content during the culturing period. One of them is seen in Fig. 1A. In other blots, the cell contour cannot be seen at all. These cells almost completely released their PACAP content (Fig. 1B – D). Further, there were seen PACAP-immunoreactive cells with sharp contour without any blots around them indicating that they did not release PACAP at all (Fig. 1B – D).
To study the effect of PACAP on LH release in pituitary cell cultures, the experiments were carried out in normal (untreated) and in PACAP-treated cell cultures. DAB chromogen was used for visualizing the LH-immunoreactive blots. Many LH blots were seen in the cell cultures in both male (Fig. 4A and B) and female rats (Fig. 4C and D). The cells showed a different LH releasing activity. The LH immunoreactivity in some of the blots was so intense that the cell contours could rarely be seen, indicating the high amount of LH released. In some cases, a well-visible dark cell contour was seen in the middle of the blots (Fig. 4C and D). In other cases, a sharp contour of LH-immunoreactive cells was seen without any blots around them (Fig. 4A and C). In treated and untreated cell cultures, a difference was observed in the diameter of the LH blots. After PACAP treatment, the diameter of the blots deriving from proestrous
3.2. Double labeling for PACAP and LH in individual pituitary cells In the pituitary cell cultures, double-labeled blots were observed with the use of a combination of PACAP and LH immunocytochemical methods. The released hormones were trapped by the nitrocellulose membrane forming blots around the cells. In the blots, PACAP was visualized by DAB chromogen and LH by fluorescent technique. In some
Fig. 3. PACAP content in the anterior pituitaries and in the cell culture media after 22-h incubation period. Optical absorbance was read at 490 nm by an ELISA reader. Standards are indicated by black columns. PACAP is present in a similar concentration in the anterior pituitaries and in the cell culture media.
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Fig. 4. LH blots deriving from male (A and B) and proestrous female rats (C and D). The cultures showed in B and D were treated with PACAP. LH immunostaining of the blots is so intense that the cell contour in the middle of the blots is rarely seen. A few of them are indicated by arrows (C and D). There are a few cells which did not release LH, indicated by arrowheads (A and C). The diameter of LH blots of cultures deriving from male rats moderately decreased upon the PACAP treatment (B compared to A); however, the diameter of those deriving from female rats increased upon PACAP treatment (D compared to C). 150 .
female rats was larger (Fig. 4D) than in untreated cultures (Fig. 4C), indicating that PACAP stimulated the LH release. The diameter of the blots of male rats was smaller in PACAP-treated (Fig. 4B) than in untreated cultures (Fig. 4A), showing that PACAP decreased the LH release, although it was moderate. The changes in the LH release were indicated by the changes of the diameter of the LH
blots formed around the cells. Image analysis of the diameters of the LH blots revealed that in the cultures deriving from proestrous female rats, the stimulation was statistically significant; however, in male cultures, the depression of LH release was not significant (Fig. 5). S-EIA revealed that at the end of the culturing period, the PACAP content of the media (to which PACAP was added in a 10 7 M concentration) was only 10 10 M. It means that the effective amount of PACAP under the culturing conditions was similar to that measured in the anterior pituitary samples (Fig. 3).
4. Discussion
Fig. 5. Image analysis of LH blots from male and female cultures. PACAP treatment significantly increased the diameter of the LH blots in cultures deriving from female rats. PACAP moderately, but not significantly, decreased the diameter of the LH blots in male cell cultures. * * * p < 0.001.
PACAP, as other bioactive brain-gut peptides reviewed by Houben and Denef [19], is also synthetised by anterior pituitary cells. The presence of PACAP in the anterior pituitary was observed by RIA and immunohistochemistry [14,20]. In female rats, PACAP-immunoreactive cells were only observed in proestrous rats, not in the other stages of the estrous cycle [14]. The expression of PACAP was also demonstrated using RT-PCR technique. With the use of the latter technique, it was shown that the expression of PACAP during the LH surge increased in cyclic female rats. The authors concluded that the steroid status influenced the PACAP expression in the anterior pituitary gland [21].
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In the present work, it has been demonstrated that PACAP is released from the cultured individual pituitary cells of both male and female rats. It was also found that there were cells that released both PACAP and LH indicated by double-labeled blots. The number of PACAP blots was very limited compared to the number of LH blots. The diameter of the PACAP blots was smaller than the diameter of the LH blots. If we accept that the diameter of the blots around the cells, demonstrated by CIBA, depends on the amount of released hormones, it becomes evident that the amount of released PACAP by a single cell is lower than that of LH. This phenomenon supports the view that LH, released in a high amount, acts in an endocrine manner on peripheral target organs reaching them through the general circulation, and PACAP, secreted in a small concentration, acts in an auto- and paracrine manner on the same or adjacent cells [22]. For studying the effect of PACAP, we compared the level of PACAP that effectively influenced the LH release in cell cultures with the level of PACAP present in the anterior pituitary under physiological conditions. By the end of the incubation period in the cell culture media, to which PACAP was added in 10 7 M concentration, the concentration of PACAP decreased to 10 10 M. The major part of PACAP added to the medium was adsorbed to the wall of the polystyrene cell culture plate during the incubation period, leaving an effective concentration of 10 10 M. The concentration of the regained peptide from the cell culture media was similar to the concentration of PACAP present in the anterior pituitary under physiological conditions. In our in vitro study, PACAP added to the cell culture medium increased the LH release from proestrous female gonadotropes, which was statistically significant. PACAP depressed the LH release from the male pituitary gonadotropes, although it was not statistically significant, as it is seen in Fig. 3. In this model, the cells were separated from each other. In an in vivo model, Leonhardt et al. [3] demonstrated an increased LH release in male rats after systematic infusion of PACAP. This phenomenon indicates that the effect of PACAP in the pituitary gland depends on other factors probably produced by other cell types present in the in loco anterior pituitary. Nagy et al. [23] have also observed that in vivo i.v. administration of PACAP to lactating mothers, separated from their pups, stimulated the prolactin release; however, in cell cultures, PACAP rather had an inhibitory effect. Jarry et al. [24] have found a contrasting effect of PACAP on prolactin and growth hormone release in male rats depending on whether an in vivo or an in vitro model was used. It has been concluded that PACAP in vivo is present in the anterior pituitary cells of rats in an amount that can influence the LH release. The effect of PACAP on LH release was stimulatory in female rats and moderately inhibitory in male rats. It is not excluded that under physiological conditions the effect of PACAP on LH release is not only direct action on the gonadotropes, but it may be
mediated through other factors as well. It is probable because PACAP receptors were demonstrated on other pituitary cell types, mainly on follicular cells and in decreasing numbers on PRL, GH, ACTH, LH, FSH and TSH cells [25]. In our model, the cells were separated from each other and the paracrine effects could not be effective.
Acknowledgements We are grateful to Mrs. Judith Fogarasi and Mrs. Anna Taka´cs for their technical assistance, and to Ms. Beata Ura´k for the computer assistance. This work was supported by ´ N, by FKFP grant AKP grant 96/2-619 3,2/56 for KK and A 1208/97 for Imre Ola´h, the head of Department of Human ´ N. Morphology, and OTKA grant T034429 for KK and A
References [1] Miyata A, Arimura A, Dahl RR, Minamino N, Uehara A, Gottschall PE, et al. Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in the pituitary cells. BBRC 1989;164:567 – 74. [2] Miyata A, Jiang L, Dahl RR, Kitada C, Kubo K, Fujino M, et al. Isolation of a neuropeptide corresponding to the N-terminal 27 residues of the pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38). BBRC 1990;170:643 – 8. [3] Leonhardt S, Jarry H, Kreipe A, Werstler K, Wuttke W. Pituitary adenylate cyclase activating polypeptide (PACAP) stimulates pituitary hormone release in male rats. Neuroendocrinol Lett 1992;14:319 – 27. [4] Sawangjaroen K, Curlewis JD. Effects of pituitary adenylate cyclaseactivating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) on prolactin, luteinizing hormone and growth hormone secretion in the ewe. J Neuroendocrinol 1994;6:549 – 55. [5] Ko¨ves K, Molna´r J, Ka´ntor O, Lakatos A, Go¨rcs TJ, Somogyva´riVı´gh A, et al. PACAP participates in the regulation of the hormonal events preceding the ovulation. Acta Biol Sci Hung 1996;47:239 – 49. [6] Ka´ntor O, Molna´r J, Heinzlmann A, Arimura A, Fu¨rst Zs, Ko¨ves K. Study on the hypothalamic factors mediating the inhibitory effect of PACAP38 on ovulation. Peptides 2002;22:2163 – 8. [7] Ka´ntor O, Molna´r J, Arimura A, Ko¨ves K. PACAP38 and PACAP27 administered intracerebroventricularly have an opposite effect on LH secretion. Peptides 2000;21:817 – 20. [8] Tsujii T, Winters SJ. Effects of pulsatile pituitary adenylate cyclase activating polypeptide (PACAP) on gonadotropin secretion and subunit mRNA levels in perifused rat pituitary cells. Life Sci 1995;56: 1103 – 11. [9] Culler MD, Paschall SC. Pituitary adenylate cyclase activating polypeptide (PACAP) potentiates the gonadotropin-releasing activity of luteinizing hormone-releasing hormone. Endocrinology 1991;129: 2260 – 2. [10] Ko¨ves K, Arimura A, Go¨rcs TJ, Somogyva´ri-Vı´gh A. Comparative distribution of immunoreactive pituitary adenylate cyclase activating polypeptide and vasoactive intestinal polypeptide in rat forebrain. Neuroendocrinology 1991;54:159 – 69. [11] Ko¨ves K, Go¨rcs TJ, Kausz M, Arimura A. Present status of the knowledge about the distribution and colocalization of PACAP in the forebrain. Acta Biol Hung 1994;45:297 – 321. [12] Ko¨ves K, Lakatos A, Somogyva´ri-Vı´gh A, Fo´gel K, Ka´ntor O, Go¨on z P, et al. Effect of the lack of light impulses on the hypothalamic PACAP and c-fos immunoreactivities in rats. Neurobiology 1997;5: 389 – 402.
E. Szabo´ et al. / Regulatory Peptides 109 (2002) 75–81 [13] Dow RC, Bennie F, Fink G. Pituitary adenylate cyclase-activating peptide-38 (PACAP-38) is released into hypophyseal portal blood in the normal male and female rats. J Endocrinol 1994;142:R1 – 4. [14] Ko¨ves K, Ka´ntor O, Scammell JG, Arimura A. PACAP colocalizes with luteinizing and follicle-stimulating hormone immunoreactivities in the anterior lobe of the pituitary gland. Peptides 1998;19:1069 – 72. [15] Arita J, Kojima Y, Kimura F. Measurement of the secretion of a small peptide at the single cell level by the cell immunoblot assay: thyroidectomy increases the number of substance P-secreting anterior pituitary cells. Endocrinology 1993;132:2682 – 8. [16] Cimini V, Van Noorden S, Mahadeva H, Polak JM. The cell blot assay in analysis of rat anterior pituitary cell secretion. Histochem J 1994;6: 59 – 66. [17] Ko¨ves K, Arimura A, Vı´gh S, Somogyva´ri-Vı´gh A, Miller J. Immunohistochemical demonstration of a novel peptide, pituitary adenylate cyclase activating polypeptide, in the ovine hypothalamus. Endocrinology 1990;127:264 – 71. [18] Suzuki N, Harada M, Kitada C, Ohkubo S, Matsumoto H, Watanabe T, et al. Production of immunoreactive pituitary adenylate cyclase activating polypeptide (PACAP) by human neuroblastoma cells, IMR-32: detection and characterization with monoclonal and polyclonal antibodies against different epitopes of PACAP. J Biochem 1993; 13:549 – 56. [19] Houben H, Denef C. Bioactive peptides in anterior pituitary cells. Peptides 1994;15:547 – 82.
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[20] Arimura A, Somogyva´ri-Vı´gh A, Miyata A, Mizuno K, Coy DH, Kitada C. Tissue distribution of PACAP as determined by RIA: highly abundant in the rat brain and testes. Endocrinology 1991;129: 2778 – 89. [21] Wuttke W, Benter S, Jarry H. Evidence for a steroid modulated expression of pituitary adenylate cyclase activating polypeptide (PACAP) in the anterior pituitary of rat. Neuroendocrinology 1994; 60(S1):17. [22] Radleff-Schlimme A, Leonhardt S, Wuttke W, Jarry H. Evidence for PACAP to be an autocrine factor on gonadotrope cells. Ann NY Acad Sci 1998;865:486 – 91. [23] Nagy Gy, Vigh S, Arimura A. PACAP induces prolactin and growth hormone release in lactating rats separated from their pups. Endocr J 1993;1:169 – 73. [24] Jarry H, Leonhardt S, Schmidt WE, Creutzfeldt W, Wuttke W. Contrasting effects of pituitary adenylate cyclase activating polypeptide (PACAP) on in vivo and in vitro prolactin and growth hormone release in male rats. Life Sci 1992;51:823 – 30. [25] Vı´gh S, Arimura A, Gottschall PE, Kitada C, Somogyva´ri-Vı´gh A, Childs GV. Cytochemical characterization of anterior pituitary target cells for the neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP), using biotinylated ligands. Peptides 1993;14: 59 – 65.
Cell immunoblot assay study demonstrating the release of PACAP from individual anterior pituitary cells of rats and the effect of PACAP on LH release ´ . Nemeske´ri a, A. Heinzlmann a, N. Suzuki b, A. Arimura c, K. Ko¨ves a,* E. Szabo´ a, A a
Department of Human Morphology and Developmental Biology, Faculty of Medicine, Semmelweis University, Tuzolto u.58, H-1094 Budapest, Hungary b Discovery Research Laboratories, Takeda Chemical Industries, Ltd., Tsukuba, Japan c US-Japan Biomedical Research Laboratories, Tulane University Hebert Center, Belle Chasse and Department of Physiology, Tulane University Medical School, New Orleans, LA, USA
Abstract The presence of pituitary adenylate cyclase activating polypeptide (PACAP) was previously demonstrated in the anterior pituitary by radioimmunoassay, immunohistochemistry, and reverse transcript-polymerase chain reaction (RT-PCR). With the use of cell immunoblot assay (CIBA), when the pituitary cells were cultured on nitrocellulose membrane, the release of PACAP by individual anterior pituitary cells was observed. The released peptide, trapped by the nitrocellulose membrane forming a blot around the cells, was demonstrated by immunocytochemistry. Double labeling revealed that a part of PACAP-immunoreactive cells can release LH as well. With the use of sandwich enzyme immunoassay (S-EIA), it was found that the concentration of PACAP in the anterior pituitaries is 10 10 M. In cell culture in a similar concentration, PACAP stimulated the LH release from female gonadotropes, but did not influence it from male ones. The stimulated release of LH was indicated by the enhancement in the diameter of LH blots compared to the untreated control cultures. We concluded that PACAP may be released from the anterior pituitary cells in a concentration which would be able to influence LH release not only in vitro but under in vivo conditions as well. The effect of PACAP on LH release was different in female and male pituitary cultures. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Immunocytochemistry; Cell immunoblot assay (CIBA); Sandwich enzyme immunoassay (S-EIA); Anterior pituitary; Rat
1. Introduction Pituitary adenylate cyclase activating polypeptide (PACAP) was isolated on the basis of its ability to stimulate adenylate cyclase in primary anterior pituitary cell cultures [1]. PACAP is present in two bioactive amidated forms, PACAP38 and PACAP27, with 38 and 27 amino acid residues, respectively [2]. It was also demonstrated that PACAP stimulates the release of several anterior pituitary hormones, among them LH, although the results published in various papers were not consistent. The models in which the effect of PACAP was studied are very different. In vivo the effect of PACAP on LH release depends on the form of PACAP (whether PACAP38 or PACAP27 *
Corresponding author. Tel.: +36-1-215-6920x3618; fax: +36-1-2153064. E-mail address: [email protected] (K. Ko¨ves).
was administered), on the species, on the dose and on the route of the administration. PACAP38 administered in an intravenous (i.v.) infusion to male rats, stimulates LH secretion [3]. When PACAP38 was given intraarterially to ovariectomized ewes, it had no effect; however, its intracerebroventricular (i.c.v.) administration inhibited LH pulse frequency and amplitude [4]. Before the critical period of the proestrous stage, i.v. administration of PACAP38 to female rats did not influence the ovulation; however, its i.c.v. administration inhibited the expected ovulation [5,6]. In the same model, i.c.v. administration of PACAP27 did not inhibit the ovulation, but enhanced the proestrous LH surge and the number of expelled ova [7]. In vitro in a static primary anterior pituitary cell culture, PACAP38 did not influence the LH release, although in a superfusion system there was a mild stimulation [1]. In perifused rat anterior pituitary cells deriving from orchid-
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ectomized male rats, PACAP38 enhanced the LH release when it was added to the medium in a pulsatile pattern [8]. In a gonadotrop cell line, PACAP38 potentiated the effect of LHRH on LH release [9]. The source of PACAP in living organism may be the hypothalamus and the anterior pituitary itself. Not in intact rats, but under special circumstances (hypophysectomy, pituitary stalk section, or the removal of the eyes), we have observed the presence of PACAP-immunoreactive fibers in the external zone of the median eminence (ME) [10 – 12]. It was also demonstrated that PACAP was released into the portal blood both in male and female rats [13]. In our laboratory, it was observed that PACAP immunoreactivity is also present in the anterior pituitary of male rats. Double labeling revealed that PACAP colocalized with LH and FSH immunoreactivities in pituitary sections. In female rats, PACAP-immunoreactive cells could only be stained when the animals were sacrificed on the day of proestrous [14]. These findings suggest that the pituitary-born PACAP, similarly to the hypothalamic one [5– 7], is also involved in the regulation of the gonadotrop hormone secretion. In the present experiment, we have studied the role of PACAP in LH secretion of male and proestrous female anterior pituitaries. The aim of our work was to investigate:
whether our method, the cell immunoblot assay, is suitable for demonstrating the PACAP release of individual anterior pituitary cells, whether PACAP-immunoreactive cells can release LH as well (it was examined by CIBA and double-labeling immunocytochemistry), what the physiological concentration of PACAP is in the anterior pituitary (it was measured with the use of sandwich enzyme immunoassay [S-EIA]), how and in what concentration PACAP influences LH release from the gonadotropes of male or female pituitaries and whether it is similar to the physiological one (it was examined by CIBA and analysed by the Image ProPlus program).
2. Materials and methods 2.1. Animals Adult (3– 4 months old) Sprague – Dawley (CRL: CD) male and proestrous female rats were used for the experiments. The animals were kept in a temperature- (22 F 2 jC) and light-controlled (lights on at 05:00 and lights off at 19:00) vivarium. The male rats were randomly chosen. Only those female rats were used for the experiments which showed regular ovarian cyclicity. Treating the animals was in accordance with the rules of the ‘‘European convention for the protection of vertebrate animals used
for experimental and other scientific purposes’’, Strasbourg, 1986. 2.2. Cell immunoblot assay (CIBA) 2.2.1. Cell culturing CIBA was carried out according to Arita et al. [15] and Cimini et al. [16] modified by one of us (Nemeske´ri). This method is suitable to demonstrate the hormone release of individual cells. Two male and two proestrous female rats were decapitated, the pituitaries were removed immediately under sterile conditions and dispersed in freshly prepared trypsin solution (Sigma-T4799) containing 0.11% trypsin in minimum essential medium (MEM) – Eagle (GIBCO EUROPE) and 0.1% bovine serum albumin (BSA). The dispersion was carried out in a Spinner flask (Bellco). We obtained cell yields of approximately 1.8 –2.4 106 cells/ pituitary of rats, with a cell viability of more than 90%. The viability of the cells was checked by vital staining. Pieces of 5 5 mm supported nitrocellulose membranes (BIO-RAD Laboratories) were placed on the surface of a metal grid in tissue culture dish (Greiner Labortechnik— 35 mm, sterile) containing 650 Al of Dulbecco-MEM (DMEM) (GIBCO BRL). Seven thousand monodispersed pituitary cells in a drop of 15 Al DMEM were placed onto the nitrocellulose membranes and cultured in 5% CO2 – 95% air incubator for 22 h. During the culturing period, the released hormones and peptides were trapped by the nitrocellulose membrane giving a possibility for immunostaining (see below). To a half of the wells containing cultured cells, PACAP was added in 10 7 M concentration. The concentration of PACAP in the media at the end of the culturing was measured by S-EIA in those wells to which PACAP was added (see later). 2.2.2. Immunocytochemistry (ICC) After the incubation period, the nitrocellulose membranes were fixed in buffered 4% paraformaldehyde solution. Those membranes that were cultured in DMEM without PACAP were immunostained for PACAP or LH. Those that were treated with PACAP were only stained for LH. The immunostaining was carried out according to the directions of the Vectastain ABC KIT (Vector Laboratories, Burlingame, CA). Antibody binding sites were visualized using H2O2 and diaminobenzidine-tetrahydrochloride (DAB). PACAP antiserum was raised in rabbit and previously characterized [14,17]. LH antiserum was raised in guinea pig and provided by NIDDK and NHPP (University of Maryland, School of Medicine, Baltimore, MD). The dilution of the PACAP antiserum was 1:6000, and that of the LH antiserum was 1:10 000. Selected membranes demonstrating PACAP blots were also stained for LH immunoreactivity using an indirect immunofluorescent method and FITC conjugate. In this latter case, the LH antiserum was used at 1:500 dilution.
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2.3. Image analysis In randomly chosen 10 viewfields of cell cultures deriving from two male and two female animals, the number and the diameter of PACAP and LH blots were analysed with the help of the Image ProPlus program (Media Cybernetics, USA). Statistical significance was calculated by the proposal of the Prizma Program. 2.4. Sandwich (two-site) enzyme immunoassay (S-EIA) 2.4.1. Preparation of the pituitary samples Fifteen male rats were sacrificed by decapitation. The pituitaries were removed immediately. The anterior pituitaries were separated, the intermediate and the posterior lobes were discarded. The anterior pituitaries were frozen on dry ice and stored at 70 jC. Before the S-EIA, the pituitaries were thawed. Three pituitaries were pooled and homogenized by ultrasound homogenizer (Ultrasonic Homogenizer, Chicago) in trifluoroacetic acid (TFA). The homogenates were centrifuged at 12 000 rpm for 20 min at 4 jC. The supernatants were dried using speed vac, then washed and dried one more time to remove TFA. The PACAP content of the samples was measured. 2.4.2. Cell culture media The PACAP content of those cell culture media to which PACAP was added was also measured at the end of the incubation period.
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2.4.3. S-EIA protocol The standard peptide (PACAP38) and the samples were put in the wells of a PA-6N (monoclonal antibody against N-terminal portion of PACAP38)-coated microtest ELISA plate and incubated for 96 h at 4 jC. The wells were washed in PBS (phosphate buffer, 0.02 M, pH 7, containing 1% bovine serum albumine). At the end of the incubation period, the wells were reacted with HRP-labeled PA-2C (monoclonal antibody against the C-terminal portion of PACAP38) for 24 h at 4 jC and were washed in PBS. The activity of HRP, bound on each well, was measured with the TMB microwell peroxidase system (Kirkegaad & Perry Labs, USA). The optical absorbance was read at 490 nm by an ELISA reader. The monoclonal antibodies against PACAP38 (PA-6N and PA-2C) were raised and tested in Suzuki’s laboratory [18].
3. Results 3.1. Release of PACAP from individual anterior pituitary cells In the cell cultures with the use of CIBA, PACAPimmunoreactive cells were observed, a few of which released PACAP. The immunoreactive PACAP in cultures were visualized by DAB chromogen. The PACAP-immunoreactive cells exhibited different releasing activity in both male and proestrous female cultures (Fig. 1). The released
Fig. 1. PACAP blots deriving from male (A and B) and proestrous female rats (C and D). In A, a PACAP-immunoreactive cell shows a sharp contour in the middle of a blot (indicated by arrow). In B, C and D, arrowheads indicate blots in which the cell contour cannot be recognized and arrows show PACAPimmunoreactive cells which did not release PACAP. 300 .
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of the double-labeled blots, a bright fluorescent halo indicated the release of LH. In the middle of these blots, the dark DAB reaction product with an irregular contour indicated the released PACAP. The amount of the released PACAP was moderate compared to the LH released by the same gonadotropes. One of them is seen in Fig. 2A. In other double-labeled blots in the middle of the bright fluorescent halo, the contour of PACAP immunoreactivity was sharp, which indicated that these gonadotropes did not release its PACAP content. One of them is seen in Fig. 2B. 3.3. PACAP content in the anterior pituitaries To study the physiological concentration of PACAP in the anterior pituitary, S-EIA was used. The level of PACAP in the anterior pituitaries was measurable. In the pooled pituitary samples, PACAP was present in 10 10 M concentration (Fig. 3). 3.4. Effect of PACAP on LH release by individual gonadotropes
Fig. 2. Double labeling for PACAP and LH in pituitary cell cultures. In A, both PACAP (dark irregular DAB reaction product, arrowhead) and LH (bright fluorescent halo, arrow) blots can be seen. This cell released a moderate amount of PACAP and a larger amount of LH. In B, the sharp contour of PACAP-immunoreactive cell (dark DAB reaction product, arrowhead) can be seen in the middle of the LH blot (bright fluorescent halo, arrow) indicating that PACAP was synthetised but not released by this cell. 900 .
PACAP formed blots around the cells. There were blots in which the contour of the cell was relatively sharp indicating that these cells did not completely release their PACAP content during the culturing period. One of them is seen in Fig. 1A. In other blots, the cell contour cannot be seen at all. These cells almost completely released their PACAP content (Fig. 1B – D). Further, there were seen PACAP-immunoreactive cells with sharp contour without any blots around them indicating that they did not release PACAP at all (Fig. 1B – D).
To study the effect of PACAP on LH release in pituitary cell cultures, the experiments were carried out in normal (untreated) and in PACAP-treated cell cultures. DAB chromogen was used for visualizing the LH-immunoreactive blots. Many LH blots were seen in the cell cultures in both male (Fig. 4A and B) and female rats (Fig. 4C and D). The cells showed a different LH releasing activity. The LH immunoreactivity in some of the blots was so intense that the cell contours could rarely be seen, indicating the high amount of LH released. In some cases, a well-visible dark cell contour was seen in the middle of the blots (Fig. 4C and D). In other cases, a sharp contour of LH-immunoreactive cells was seen without any blots around them (Fig. 4A and C). In treated and untreated cell cultures, a difference was observed in the diameter of the LH blots. After PACAP treatment, the diameter of the blots deriving from proestrous
3.2. Double labeling for PACAP and LH in individual pituitary cells In the pituitary cell cultures, double-labeled blots were observed with the use of a combination of PACAP and LH immunocytochemical methods. The released hormones were trapped by the nitrocellulose membrane forming blots around the cells. In the blots, PACAP was visualized by DAB chromogen and LH by fluorescent technique. In some
Fig. 3. PACAP content in the anterior pituitaries and in the cell culture media after 22-h incubation period. Optical absorbance was read at 490 nm by an ELISA reader. Standards are indicated by black columns. PACAP is present in a similar concentration in the anterior pituitaries and in the cell culture media.
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Fig. 4. LH blots deriving from male (A and B) and proestrous female rats (C and D). The cultures showed in B and D were treated with PACAP. LH immunostaining of the blots is so intense that the cell contour in the middle of the blots is rarely seen. A few of them are indicated by arrows (C and D). There are a few cells which did not release LH, indicated by arrowheads (A and C). The diameter of LH blots of cultures deriving from male rats moderately decreased upon the PACAP treatment (B compared to A); however, the diameter of those deriving from female rats increased upon PACAP treatment (D compared to C). 150 .
female rats was larger (Fig. 4D) than in untreated cultures (Fig. 4C), indicating that PACAP stimulated the LH release. The diameter of the blots of male rats was smaller in PACAP-treated (Fig. 4B) than in untreated cultures (Fig. 4A), showing that PACAP decreased the LH release, although it was moderate. The changes in the LH release were indicated by the changes of the diameter of the LH
blots formed around the cells. Image analysis of the diameters of the LH blots revealed that in the cultures deriving from proestrous female rats, the stimulation was statistically significant; however, in male cultures, the depression of LH release was not significant (Fig. 5). S-EIA revealed that at the end of the culturing period, the PACAP content of the media (to which PACAP was added in a 10 7 M concentration) was only 10 10 M. It means that the effective amount of PACAP under the culturing conditions was similar to that measured in the anterior pituitary samples (Fig. 3).
4. Discussion
Fig. 5. Image analysis of LH blots from male and female cultures. PACAP treatment significantly increased the diameter of the LH blots in cultures deriving from female rats. PACAP moderately, but not significantly, decreased the diameter of the LH blots in male cell cultures. * * * p < 0.001.
PACAP, as other bioactive brain-gut peptides reviewed by Houben and Denef [19], is also synthetised by anterior pituitary cells. The presence of PACAP in the anterior pituitary was observed by RIA and immunohistochemistry [14,20]. In female rats, PACAP-immunoreactive cells were only observed in proestrous rats, not in the other stages of the estrous cycle [14]. The expression of PACAP was also demonstrated using RT-PCR technique. With the use of the latter technique, it was shown that the expression of PACAP during the LH surge increased in cyclic female rats. The authors concluded that the steroid status influenced the PACAP expression in the anterior pituitary gland [21].
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In the present work, it has been demonstrated that PACAP is released from the cultured individual pituitary cells of both male and female rats. It was also found that there were cells that released both PACAP and LH indicated by double-labeled blots. The number of PACAP blots was very limited compared to the number of LH blots. The diameter of the PACAP blots was smaller than the diameter of the LH blots. If we accept that the diameter of the blots around the cells, demonstrated by CIBA, depends on the amount of released hormones, it becomes evident that the amount of released PACAP by a single cell is lower than that of LH. This phenomenon supports the view that LH, released in a high amount, acts in an endocrine manner on peripheral target organs reaching them through the general circulation, and PACAP, secreted in a small concentration, acts in an auto- and paracrine manner on the same or adjacent cells [22]. For studying the effect of PACAP, we compared the level of PACAP that effectively influenced the LH release in cell cultures with the level of PACAP present in the anterior pituitary under physiological conditions. By the end of the incubation period in the cell culture media, to which PACAP was added in 10 7 M concentration, the concentration of PACAP decreased to 10 10 M. The major part of PACAP added to the medium was adsorbed to the wall of the polystyrene cell culture plate during the incubation period, leaving an effective concentration of 10 10 M. The concentration of the regained peptide from the cell culture media was similar to the concentration of PACAP present in the anterior pituitary under physiological conditions. In our in vitro study, PACAP added to the cell culture medium increased the LH release from proestrous female gonadotropes, which was statistically significant. PACAP depressed the LH release from the male pituitary gonadotropes, although it was not statistically significant, as it is seen in Fig. 3. In this model, the cells were separated from each other. In an in vivo model, Leonhardt et al. [3] demonstrated an increased LH release in male rats after systematic infusion of PACAP. This phenomenon indicates that the effect of PACAP in the pituitary gland depends on other factors probably produced by other cell types present in the in loco anterior pituitary. Nagy et al. [23] have also observed that in vivo i.v. administration of PACAP to lactating mothers, separated from their pups, stimulated the prolactin release; however, in cell cultures, PACAP rather had an inhibitory effect. Jarry et al. [24] have found a contrasting effect of PACAP on prolactin and growth hormone release in male rats depending on whether an in vivo or an in vitro model was used. It has been concluded that PACAP in vivo is present in the anterior pituitary cells of rats in an amount that can influence the LH release. The effect of PACAP on LH release was stimulatory in female rats and moderately inhibitory in male rats. It is not excluded that under physiological conditions the effect of PACAP on LH release is not only direct action on the gonadotropes, but it may be
mediated through other factors as well. It is probable because PACAP receptors were demonstrated on other pituitary cell types, mainly on follicular cells and in decreasing numbers on PRL, GH, ACTH, LH, FSH and TSH cells [25]. In our model, the cells were separated from each other and the paracrine effects could not be effective.
Acknowledgements We are grateful to Mrs. Judith Fogarasi and Mrs. Anna Taka´cs for their technical assistance, and to Ms. Beata Ura´k for the computer assistance. This work was supported by ´ N, by FKFP grant AKP grant 96/2-619 3,2/56 for KK and A 1208/97 for Imre Ola´h, the head of Department of Human ´ N. Morphology, and OTKA grant T034429 for KK and A
References [1] Miyata A, Arimura A, Dahl RR, Minamino N, Uehara A, Gottschall PE, et al. Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in the pituitary cells. BBRC 1989;164:567 – 74. [2] Miyata A, Jiang L, Dahl RR, Kitada C, Kubo K, Fujino M, et al. Isolation of a neuropeptide corresponding to the N-terminal 27 residues of the pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38). BBRC 1990;170:643 – 8. [3] Leonhardt S, Jarry H, Kreipe A, Werstler K, Wuttke W. Pituitary adenylate cyclase activating polypeptide (PACAP) stimulates pituitary hormone release in male rats. Neuroendocrinol Lett 1992;14:319 – 27. [4] Sawangjaroen K, Curlewis JD. Effects of pituitary adenylate cyclaseactivating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) on prolactin, luteinizing hormone and growth hormone secretion in the ewe. J Neuroendocrinol 1994;6:549 – 55. [5] Ko¨ves K, Molna´r J, Ka´ntor O, Lakatos A, Go¨rcs TJ, Somogyva´riVı´gh A, et al. PACAP participates in the regulation of the hormonal events preceding the ovulation. Acta Biol Sci Hung 1996;47:239 – 49. [6] Ka´ntor O, Molna´r J, Heinzlmann A, Arimura A, Fu¨rst Zs, Ko¨ves K. Study on the hypothalamic factors mediating the inhibitory effect of PACAP38 on ovulation. Peptides 2002;22:2163 – 8. [7] Ka´ntor O, Molna´r J, Arimura A, Ko¨ves K. PACAP38 and PACAP27 administered intracerebroventricularly have an opposite effect on LH secretion. Peptides 2000;21:817 – 20. [8] Tsujii T, Winters SJ. Effects of pulsatile pituitary adenylate cyclase activating polypeptide (PACAP) on gonadotropin secretion and subunit mRNA levels in perifused rat pituitary cells. Life Sci 1995;56: 1103 – 11. [9] Culler MD, Paschall SC. Pituitary adenylate cyclase activating polypeptide (PACAP) potentiates the gonadotropin-releasing activity of luteinizing hormone-releasing hormone. Endocrinology 1991;129: 2260 – 2. [10] Ko¨ves K, Arimura A, Go¨rcs TJ, Somogyva´ri-Vı´gh A. Comparative distribution of immunoreactive pituitary adenylate cyclase activating polypeptide and vasoactive intestinal polypeptide in rat forebrain. Neuroendocrinology 1991;54:159 – 69. [11] Ko¨ves K, Go¨rcs TJ, Kausz M, Arimura A. Present status of the knowledge about the distribution and colocalization of PACAP in the forebrain. Acta Biol Hung 1994;45:297 – 321. [12] Ko¨ves K, Lakatos A, Somogyva´ri-Vı´gh A, Fo´gel K, Ka´ntor O, Go¨on z P, et al. Effect of the lack of light impulses on the hypothalamic PACAP and c-fos immunoreactivities in rats. Neurobiology 1997;5: 389 – 402.
E. Szabo´ et al. / Regulatory Peptides 109 (2002) 75–81 [13] Dow RC, Bennie F, Fink G. Pituitary adenylate cyclase-activating peptide-38 (PACAP-38) is released into hypophyseal portal blood in the normal male and female rats. J Endocrinol 1994;142:R1 – 4. [14] Ko¨ves K, Ka´ntor O, Scammell JG, Arimura A. PACAP colocalizes with luteinizing and follicle-stimulating hormone immunoreactivities in the anterior lobe of the pituitary gland. Peptides 1998;19:1069 – 72. [15] Arita J, Kojima Y, Kimura F. Measurement of the secretion of a small peptide at the single cell level by the cell immunoblot assay: thyroidectomy increases the number of substance P-secreting anterior pituitary cells. Endocrinology 1993;132:2682 – 8. [16] Cimini V, Van Noorden S, Mahadeva H, Polak JM. The cell blot assay in analysis of rat anterior pituitary cell secretion. Histochem J 1994;6: 59 – 66. [17] Ko¨ves K, Arimura A, Vı´gh S, Somogyva´ri-Vı´gh A, Miller J. Immunohistochemical demonstration of a novel peptide, pituitary adenylate cyclase activating polypeptide, in the ovine hypothalamus. Endocrinology 1990;127:264 – 71. [18] Suzuki N, Harada M, Kitada C, Ohkubo S, Matsumoto H, Watanabe T, et al. Production of immunoreactive pituitary adenylate cyclase activating polypeptide (PACAP) by human neuroblastoma cells, IMR-32: detection and characterization with monoclonal and polyclonal antibodies against different epitopes of PACAP. J Biochem 1993; 13:549 – 56. [19] Houben H, Denef C. Bioactive peptides in anterior pituitary cells. Peptides 1994;15:547 – 82.
81
[20] Arimura A, Somogyva´ri-Vı´gh A, Miyata A, Mizuno K, Coy DH, Kitada C. Tissue distribution of PACAP as determined by RIA: highly abundant in the rat brain and testes. Endocrinology 1991;129: 2778 – 89. [21] Wuttke W, Benter S, Jarry H. Evidence for a steroid modulated expression of pituitary adenylate cyclase activating polypeptide (PACAP) in the anterior pituitary of rat. Neuroendocrinology 1994; 60(S1):17. [22] Radleff-Schlimme A, Leonhardt S, Wuttke W, Jarry H. Evidence for PACAP to be an autocrine factor on gonadotrope cells. Ann NY Acad Sci 1998;865:486 – 91. [23] Nagy Gy, Vigh S, Arimura A. PACAP induces prolactin and growth hormone release in lactating rats separated from their pups. Endocr J 1993;1:169 – 73. [24] Jarry H, Leonhardt S, Schmidt WE, Creutzfeldt W, Wuttke W. Contrasting effects of pituitary adenylate cyclase activating polypeptide (PACAP) on in vivo and in vitro prolactin and growth hormone release in male rats. Life Sci 1992;51:823 – 30. [25] Vı´gh S, Arimura A, Gottschall PE, Kitada C, Somogyva´ri-Vı´gh A, Childs GV. Cytochemical characterization of anterior pituitary target cells for the neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP), using biotinylated ligands. Peptides 1993;14: 59 – 65.