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Fos activation and upregulation of nicotinamide adenine dinucleotide phosphate diaphorase in the rat pituitary by acute capsaicin injection
Neuroscience Letters 295 (2000) 73±76
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Fos activation and upregulation of nicotinamide adenine dinucleotide phosphate diaphorase in the rat pituitary by acute capsaicin injection Chuma O. Okere a,*, Takashi Higuchi b, Hideto Kaba a a
Department of Physiology, Kochi Medical School, Okoh-cho, Nankoku, Kochi 783±8505, Japan b Department of Physiology, Fukui Medical University, Matsuoka, Fukui 910±1193, Japan Received 9 September 2000; accepted 21 September 2000
Abstract In this study, we examined the effect of acute capsaicin injection on nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d, a histochemical index of nitric oxide, NO, synthase) and Fos expression in the rat pituitary gland. Compared with vehicle, capsaicin signi®cantly activated Fos expression in the anterior and intermediate lobes. In addition, capsaicin-treated rats showed a signi®cant upregulation of NADPH-d in the anterior and neural lobes. Pretreatment of the animals with a speci®c NO synthase inhibitor, N v-nitro-l-arginine methyl ester (l-NAME), signi®cantly attenuated the capsaicin-induced Fos expression in the anterior and intermediate lobes. These observations suggest that NO is a key regulator of the acute effect of capsaicin on the pituitary gland. q 2000 Published by Elsevier Science Ireland Ltd. Keywords: Capsaicin; Fos; Nicotinamide adenine dinucleotide phosphate-diaphorase; Nitric oxide synthase; N v-nitro-l-arginine methyl ester; Pituitary
The dose and method of administration rather than the route, determine the effect of capsaicin (8-methyl-N-vanillyl-6-nonenamide, the primary pungent substance in red, hot peppers) on the nervous system. While acute capsaicin administration excites primary afferent neurons connected to distinct sensory receptors, a prolonged or long-term exposure to capsaicin causes ablation of primary afferent neurons and reorganization of capsaicin-sensitive primary afferent neurons [5]. Capsaicin activation of sensory neurons stimulates several central regulatory re¯exes including neuroendocrine control mechanisms [9,19]. The mechanism of capsaicin action partly involves N-methyl-d-aspartate (NMDA) receptor activation [16] and possibly nitric oxide (NO) [20], given that activation of the NMDA receptor is accompanied by NO synthesis and release [4]. Chronic capsaicin treatment may result in a marked reduction in sensitivity of the hypothalamo-hypophysial axis. For instance, capsaicin has been reported to selectively decrease neuronal activity (as assessed by Fos expression) in the para* Corresponding author. Present address: Department of Neurobiology and Anatomy, MCP Hahnemann University School of Medicine, 2900 Queen Lane, Philadelphia, PA 19129, USA. Tel.: 11-215-991-8428; fax: 11-215-843-9082. E-mail address: [email protected] (C.O. Okere).
ventricular nucleus after abdominal surgery [2]. Also, chemical sensory deafferentation produced by chronic capsaicin treatment abolished hypothalamic-pituitary activation by noxious stimulation or acupuncture [14]. Furthermore, in the pituitary gland, noxious heat or electroacupuncture caused signi®cantly lower Fos expression in capsaicin-sensitized than in vehicle-treated animals [14]. While there are several reports on the effect of chronic capsaicin administration on the pituitary gland, there is not much information on the effect of acute capsaicin treatment on the pituitary gland or what messenger molecules may be involved. The pituitary gland is functionally regulated by neurosecretory substances released from hypothalamic afferents into the hypophysial portal circulation via nerve terminals projecting to the arcuate nucleus and the median eminence. Recently, we reported that acute capsaicin injection signi®cantly increased nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d, a histochemical index of NO synthase, NOS) and Fos expression in the arcuate nucleus and median eminence [11]. Also, endogenous NOS is an important mediator of capsaicin effect within the paraventricular and supraoptic nuclei of the hypothalamus as well as in the amygdala [13]. Together with the hypothalamus and the adrenal gland, the pituitary gland is an important component of the
0304-3940/00/$ - see front matter q 2000 Published by Elsevier Science Ireland Ltd. PII: S03 04 - 394 0( 0 0) 01 60 9- 8
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C.O. Okere et al. / Neuroscience Letters 295 (2000) 73±76
neuroendocrine pathway which responds to challenges to the internal milieu. Therefore, the rationale for this study was to examine how acute capsaicin injection may affect cellular activity and the possible involvement of NO as a neuromodulator of the effect of capsaicin on the pituitary gland. In this report, we describe Fos and NADPH-d expression in the pituitary gland of rats after acute capsaicin injection. Two groups of male Wistar strain rats (n 4 each) weighing 250±500 g (Japan SLC, Hamamatsu, Japan) were subcutaneously injected with capsaicin (50 mg/kg body weight b.w.; Wako Pure Chem., Japan) or vehicle (8:1:1 v/v saline:alcohol:Tween 20). To examine NOS involvement in the pituitary gland response to capsaicin, a separate group of four animals each were injected with N v-nitro-l-arginine methyl ester [l-NAME, 50 mg/kg b.w., intraperitoneal (i.p.), Sigma, St. Louis, MO] or the enantiomer d-NAME 30 min before the injection of capsaicin. Forty-®ve minutes after capsaicin injection, the animals were anesthetized with Nembutal (50 mg/kg, b.w., i.p.) and then transcardially perfused with ice-cold 0.01 M phosphate buffer (PB, pH 7.3) followed by cold 4% paraformaldehyde in 0.01 M PB. The pituitary gland was harvested, post®xed in ®xative and cryoprotected in 30% sucrose in 0.01 M PB. Sections from all the animals were processed together to eliminate handling variability. The expression of NADPH-d was demonstrated by incubating free-¯oating, 50 mm-thick sections of the pituitary gland for 45 min in a medium containing 10 mg reduced bNADPH (Sigma, St. Louis, MO), 1 mg nitroblue tetrazolium (Sigma, St. Louis, MO) in 10 ml of 20 mM PB (pH 7.4) containing 0.3% Triton X-100. The reaction was stopped by rinsing the sections in PB, followed by mounting on gelatin-subbed slides, air drying, dehydration in alcohol and coverslipping. The NADPH-d reactivity was semi-quanti®ed as previously described [11]. To demonstrate Fos immunoreactivity, separate sections were processed as previously described [12] and the ®nal reaction product was intensi®ed with nickel. The Fos-positive cells were manually counted in at least two sections from each animal and the values tested statistically using the Mann±Whitney U-test and the Wilcoxon-ranked test at the 5% level of signi®cance. The anterior and posterior lobes of the pituitary, but not the intermediate lobe, showed positive NADPH-d reactivity (Fig. 1). The neural (posterior) lobe of capsaicin-treated animals showed a signi®cant upregulation of NADPH-d staining compared with vehicle-treated animals (265 ^ 33 vs. 163 ^ 27 arbitrary units per section; P , 0:01, Mann± Whitney U-test; Fig. 1a,b). Within the anterior lobe, capsaicin-treated rats showed a marked upregulation of NADPH-d compared with vehicle-treated animals (Fig. 1c,d). Capsaicin-treated animals exhibited a relatively stronger NADPH-d staining in the folliculo-stellate cells than was observed in vehicle-treated rats (arrows, Fig. 1c). Compared with d-NAME, pretreatment with l-NAME before capsaicin injection reduced the expression
of NADPH-d in the anterior and posterior lobes (P , 0:01, Wilcoxon test; Fig. 2A,B). Capsaicin activated Fos expression in the intermediate and anterior lobes. Although there was no strict topographical organization of the Fos-positive cells in the anterior lobe, there was a tendency for these cells to be predominantly localized distal to the intermediate lobe. A signi®cantly higher (P , 0:01, Mann±Whitney U-test) activation of Fos expression was observed in the anterior and intermediate lobes after capsaicin injection compared with vehicle (426 ^ 67 vs. 45 ^ 13 cells per section; Fig. 1e,f). The posterior lobe lacked Fos immunoreactive cells. Fos expression in the anterior and intermediate lobes of capsaicintreated animals was signi®cantly attenuated by pretreatment with l-NAME compared with d-NAME (236 ^ 91 vs. 399 ^ 61 cells per section; P , 0:05, Mann±Whitney Utest; Fig. 2C,D). Hence capsaicin-induced activation of cellular response in the anterior and intermediate lobes of the pituitary requires NOS mediation. This observation, however, does not preclude the involvement of other mediator molecules. The results presented in this report favor a regulatory in¯uence of NOS in the effect of capsaicin on the pituitary gland. The major ®nding in this study is capsaicin-induced activation of Fos and NADPH-d expression in the pituitary gland and attenuation of the capsaicin-induced Fos expression by pretreatment with a speci®c NOS inhibitor. The observed reduction in NADPH-d staining in the pituitary gland after l-NAME pretreatment agrees with the ability of NOS inhibitors to block NADPH-d expression. In the neural lobe, this reduction is consistent with the decreased NADPH-d staining in the cell bodies of magnocellular neurons in the hypothalamus [13]. That capsaicin activated marked Fos expression in the intermediate and anterior lobes agrees with previous ®ndings in animals stressed by immobilization [8,17]. Different types of stressors appear to activate the release of adrenocorticotropic hormone (ACTH) from either the intermediate or anterior lobes [15] and predominantly activate Fos expression in ACTH cells [13]. Although there is, as yet, no direct evidence of NOS colocalization with ACTH cells, an increased vascular supply to secretory cells resulting from the perivascular localization of NOS in the anterior lobe [10] and the property of NO as a freely diffusible neurotransmitter potentially allow for a modulation of ACTH [3] and secretion of other hormone(s). Further studies using double labeling techniques will be necessary to determine the detailed pattern of Fos and NADPH-d expression together with other regulators of stress in the anterior lobe of the pituitary gland. The principal neuroendocrine response to acute physical or psychological stress is the synthesis and secretion, by the hypothalamo-pituitary-adrenal axis, of corticotropin-releasing factor (CRF), ACTH and propiomelanocortin-derived peptides as well as corticosteroids [1]. The CRF neurons from the parvocellular paraventricular nucleus of the hypothalamus project to the median eminence where secre-
C.O. Okere et al. / Neuroscience Letters 295 (2000) 73±76
75
Fig. 1. Photomicrographs from one set of animals showing NADPH diaphorase reactivity (a±d) and Fos immunoreactive cells (e,f) in the pituitary gland of capsaicin- (a,c,e) and vehicle- (b,d,f) treated animals. The posterior (a vs. b) and anterior (c vs. d, higher magni®cation) lobes of capsaicin-treated animals showed a higher NADPH diaphorase staining. More Fos immunoreactive cells were present in the anterior and intermediate lobes in response to capsaicin (e vs. f). e2 represents a section from regions of the anterior lobe lateral to the intermediate lobe in a coronal plane. Scale bar in (f) 200 mm in (a,b,e,f); 100 mm, in (c,d). Arrows in (c) indicate folliculo-stellate cells. AL, anterior lobe; IL, intermediate lobe; PL, posterior (neural) lobe.
Fig. 2. Photomicrographs from a set of animals showing the effect of the NOS inhibitor-(l-NAME, A,C) or its (control) inactive enantiomer d-NAME (B,D) pretreatment on capsaicin-induced NADPH diaphorase (A,B) and Fos immunoreactivity (C,D) in the pituitary. Pretreatment with l-NAME attenuated the capsaicin-induced Fos expression and NADPH diaphorase in the anterior lobe. Scale bar in (D) 200 mm (A,B) and 400 mm in (C,D).
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C.O. Okere et al. / Neuroscience Letters 295 (2000) 73±76
tion of CRF into the hypophysial portal blood occurs. In a previous report, capsaicin upregulated the expression of NADPH-d in the internal zone of the median eminence and Fos expression in the arcuate nucleus [11]. The present study showed a capsaicin-induced upregulation of NADPHd in the neural lobe. Since the neural lobe is responsible for the secretion of oxytocin and vasopressin, this suggests that capsaicin may alter the secretion of either or both hormones. Interestingly, chronic capsaicin treatment has been reported to stimulate substance P release in the supraoptic nucleus and to cause antidiuresis as a result of increased release of vasopressin from the neural lobe [18]. The signi®cance of the relatively higher NADPH-d staining in the folliculostellate cells in capsaicin-treated animals observed in this study is obscure. Although NOS expression is upregulated in the hypothalamo-pituitary-adrenal axis during immobilization stress [7] and capsaicin activates immediate early gene expression in the paraventricular nucleus [6], there is no evidence to our knowledge, linking capsaicin-induced cellular activation to an upregulation of NOS activity. Taken together with previous observations of capsaicin-induced activation of NOS neurons in the arcuate nucleus, median eminence and hypothalamus [11,13], the present observations indicate that NO is a principal mediator of capsaicin effect on the hypothalamo-pituitary axis. This hypothesis is supported by the ®nding that in animals pretreated with a NOS inhibitor, the capsaicin-induced activation of Fos expression was signi®cantly attenuated. In summary, evidence reported in this study showed that capsaicin treatment caused cellular (Fos) activation and upregulation of NADPH-diaphorase (NOS) reactivity in the pituitary gland. Pretreatment with a NOS inhibitor attenuated the capsaicin-induced cellular activation. Taken together, the present observations suggest that the effect of acute capsaicin on the pituitary gland requires a signi®cant NO mediation. This work was supported by research grants from the Ministry of Education, Science, Sports and Culture of Japan. C.O. Okere was in receipt of a Japan Society for the Promotion of Science Postdoctoral Fellowship. [1] Axelrod, J. and Reisine, T.D., Stress hormones: their interaction and regulation, Science, 224 (1984) 452±459. [2] Bonaz, B. and TacheÂ, Y., Corticotropin-releasing factor and systemic capsaicin-sensitive afferents are involved in abdominal surgery-induced Fos expression in the paraventricular nucleus of the hypothalamus, Brain Res., 748 (1997) 12±20. [3] Brunetti, L., Preziosi, P., Ragazzoni, E. and Vacca, M., Involvement of nitric oxide in basal and interleukin 1b-induced CRH and ACTH release in vitro, Life Sci., 53 (1993) PL219± PL222. [4] Garthwaite, J., Garthwaite, G., Palmer, R.M.J. and Moncada, S., NMDA receptor activation induces nitric oxide synthesis from arginine in rat brain slices, Eur. J. Pharmacol., 172 (1989) 413±416.
[5] Holzer, P., Capsaicin: cellular targets, mechanisms of action and selectivity for thin sensory neurons, Pharmacol. Rev., 43 (1991) 143±201. [6] Honkaniemi, J., Kainu, T., Ceccatelli, S., Rechardt, L., HoÈkfelt, T. and Pelto-Huikko, M., Fos and jun in rat central amygdaloid nucleus and paraventricular nucleus after stress, NeuroReport, 3 (1992) 849±852. [7] Kishimoto, J., Tsuchiya, T., Emson, P.C. and Nakayama, Y., Immobilization-induced stress activates neuronal nitric oxide synthase (nNOS) mRNA and protein in hypothalamo-pituitary-adrenal axis in rats, Brain Res., 720 (1996) 159±171. [8] Kononen, J., Honkaniemi, J., Alho, H., Koistinaho, J., Iadarola, M. and Pelto-Huikko, M., Fos-like immunoreactivity in the rat hypothalamic-pituitary axis after immobilization stress, Endocrinology, 130 (1992) 3041±3047. [9] Mueller, G.P., Beta-endorphin immunoreactivity in rat plasma: variations in response to different physical stimuli, Life Sci., 29 (1981) 1669±1674. [10] Okere, C.O., Murata, E. and Higuchi, T., Perivascular localization of nitric oxide synthase in the rat adenohypophysis: potential implications for function and cell-cell interaction, Brain Res., 784 (1998) 337±340. [11] Okere, C.O., Kaba, H. and Higuchi, T., A role for capsaicin in the median eminence and arcuate nucleus response to capsaicin treatment in rats, NeuroReport, 10 (1999) 1209± 1213. [12] Okere, C.O., Kaba, H., Seto, K. and Higuchi, T., Intracerebroventricular injection of a nitric oxide donor attenuates Fos expression in the paraventricular and supraoptic nuclei of lactating rats, Brain Res., 828 (1999) 104±114. [13] Okere, C.O., Kaba, H. and Higuchi, T., Importance of endogenous nitric oxide synthase in the rat hypothalamus and amygdala in mediating the response to capsaicin, J. Comp. Neurol., 423 (2000) 670±686. [14] Pan, B., Castro-Lopez, J.M. and Coimbra, A., Chemical sensory deafferentation abolishes hypothalamic pituitary activation induced by noxious stimulation or electroacupuncture but only decreases that caused by immobilization stress. A c-Fos study, Neuroscience, 78 (1997) 1059± 1068. [15] Przewlocki, R., Millan, M.J., Gramsch, C.H., Millan, M.H. and Herz, A., The in¯uences of selective adeno- and neurointermedio-hypophysectomy upon plasma and brain levels of b-endorphin and their response to stress, Brain Res., 242 (1982) 107±117. [16] Sakurada, T., Sugiyama, A., Sakurada, C., Tanno, K., Sakurada, S., Kisara, K., Hara, A. and Abiko, Y., Involvement of nitric oxide in spinally mediated capsaicin- and glutamateinduced behavioral responses in the mouse, Neurochem. Int., 29 (1996) 271±278. [17] Senba, E., Umemoto, S., Kawai, Y. and Noguchi, K., Differential expression of fos family and jun family mRNAs in the rat hypothalamo-pituitary-adrenal axis after immobilization stress, Mol. Brain Res., 24 (1994) 283±294. [18] Tsushima, H. and Mori, M., Central injections of capsaicin cause antidiuresis mediated through neurokinin-1 receptors in rat hypothalamus and vasopressin release, Jpn. J. Pharmacol., 71 (1999) 237±241. [19] Watanabe, T., Kawada, T., Kurosawa, M., Sato, A. and Iwai, K., Adrenal sympathetic efferent nerve and catecholamine secretion excitation caused by capsaicin in rats, Am. J. Physiol., 255 (1988) E23±E27. [20] Wu, J., Lin, Q., McAdoo, D.J. and Willis, W.D., Nitric oxide contributes to central sensitization following intradermal injection of capsaicin, NeuroReport, 9 (1998) 589± 592.
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Fos activation and upregulation of nicotinamide adenine dinucleotide phosphate diaphorase in the rat pituitary by acute capsaicin injection Chuma O. Okere a,*, Takashi Higuchi b, Hideto Kaba a a
Department of Physiology, Kochi Medical School, Okoh-cho, Nankoku, Kochi 783±8505, Japan b Department of Physiology, Fukui Medical University, Matsuoka, Fukui 910±1193, Japan Received 9 September 2000; accepted 21 September 2000
Abstract In this study, we examined the effect of acute capsaicin injection on nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d, a histochemical index of nitric oxide, NO, synthase) and Fos expression in the rat pituitary gland. Compared with vehicle, capsaicin signi®cantly activated Fos expression in the anterior and intermediate lobes. In addition, capsaicin-treated rats showed a signi®cant upregulation of NADPH-d in the anterior and neural lobes. Pretreatment of the animals with a speci®c NO synthase inhibitor, N v-nitro-l-arginine methyl ester (l-NAME), signi®cantly attenuated the capsaicin-induced Fos expression in the anterior and intermediate lobes. These observations suggest that NO is a key regulator of the acute effect of capsaicin on the pituitary gland. q 2000 Published by Elsevier Science Ireland Ltd. Keywords: Capsaicin; Fos; Nicotinamide adenine dinucleotide phosphate-diaphorase; Nitric oxide synthase; N v-nitro-l-arginine methyl ester; Pituitary
The dose and method of administration rather than the route, determine the effect of capsaicin (8-methyl-N-vanillyl-6-nonenamide, the primary pungent substance in red, hot peppers) on the nervous system. While acute capsaicin administration excites primary afferent neurons connected to distinct sensory receptors, a prolonged or long-term exposure to capsaicin causes ablation of primary afferent neurons and reorganization of capsaicin-sensitive primary afferent neurons [5]. Capsaicin activation of sensory neurons stimulates several central regulatory re¯exes including neuroendocrine control mechanisms [9,19]. The mechanism of capsaicin action partly involves N-methyl-d-aspartate (NMDA) receptor activation [16] and possibly nitric oxide (NO) [20], given that activation of the NMDA receptor is accompanied by NO synthesis and release [4]. Chronic capsaicin treatment may result in a marked reduction in sensitivity of the hypothalamo-hypophysial axis. For instance, capsaicin has been reported to selectively decrease neuronal activity (as assessed by Fos expression) in the para* Corresponding author. Present address: Department of Neurobiology and Anatomy, MCP Hahnemann University School of Medicine, 2900 Queen Lane, Philadelphia, PA 19129, USA. Tel.: 11-215-991-8428; fax: 11-215-843-9082. E-mail address: [email protected] (C.O. Okere).
ventricular nucleus after abdominal surgery [2]. Also, chemical sensory deafferentation produced by chronic capsaicin treatment abolished hypothalamic-pituitary activation by noxious stimulation or acupuncture [14]. Furthermore, in the pituitary gland, noxious heat or electroacupuncture caused signi®cantly lower Fos expression in capsaicin-sensitized than in vehicle-treated animals [14]. While there are several reports on the effect of chronic capsaicin administration on the pituitary gland, there is not much information on the effect of acute capsaicin treatment on the pituitary gland or what messenger molecules may be involved. The pituitary gland is functionally regulated by neurosecretory substances released from hypothalamic afferents into the hypophysial portal circulation via nerve terminals projecting to the arcuate nucleus and the median eminence. Recently, we reported that acute capsaicin injection signi®cantly increased nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d, a histochemical index of NO synthase, NOS) and Fos expression in the arcuate nucleus and median eminence [11]. Also, endogenous NOS is an important mediator of capsaicin effect within the paraventricular and supraoptic nuclei of the hypothalamus as well as in the amygdala [13]. Together with the hypothalamus and the adrenal gland, the pituitary gland is an important component of the
0304-3940/00/$ - see front matter q 2000 Published by Elsevier Science Ireland Ltd. PII: S03 04 - 394 0( 0 0) 01 60 9- 8
74
C.O. Okere et al. / Neuroscience Letters 295 (2000) 73±76
neuroendocrine pathway which responds to challenges to the internal milieu. Therefore, the rationale for this study was to examine how acute capsaicin injection may affect cellular activity and the possible involvement of NO as a neuromodulator of the effect of capsaicin on the pituitary gland. In this report, we describe Fos and NADPH-d expression in the pituitary gland of rats after acute capsaicin injection. Two groups of male Wistar strain rats (n 4 each) weighing 250±500 g (Japan SLC, Hamamatsu, Japan) were subcutaneously injected with capsaicin (50 mg/kg body weight b.w.; Wako Pure Chem., Japan) or vehicle (8:1:1 v/v saline:alcohol:Tween 20). To examine NOS involvement in the pituitary gland response to capsaicin, a separate group of four animals each were injected with N v-nitro-l-arginine methyl ester [l-NAME, 50 mg/kg b.w., intraperitoneal (i.p.), Sigma, St. Louis, MO] or the enantiomer d-NAME 30 min before the injection of capsaicin. Forty-®ve minutes after capsaicin injection, the animals were anesthetized with Nembutal (50 mg/kg, b.w., i.p.) and then transcardially perfused with ice-cold 0.01 M phosphate buffer (PB, pH 7.3) followed by cold 4% paraformaldehyde in 0.01 M PB. The pituitary gland was harvested, post®xed in ®xative and cryoprotected in 30% sucrose in 0.01 M PB. Sections from all the animals were processed together to eliminate handling variability. The expression of NADPH-d was demonstrated by incubating free-¯oating, 50 mm-thick sections of the pituitary gland for 45 min in a medium containing 10 mg reduced bNADPH (Sigma, St. Louis, MO), 1 mg nitroblue tetrazolium (Sigma, St. Louis, MO) in 10 ml of 20 mM PB (pH 7.4) containing 0.3% Triton X-100. The reaction was stopped by rinsing the sections in PB, followed by mounting on gelatin-subbed slides, air drying, dehydration in alcohol and coverslipping. The NADPH-d reactivity was semi-quanti®ed as previously described [11]. To demonstrate Fos immunoreactivity, separate sections were processed as previously described [12] and the ®nal reaction product was intensi®ed with nickel. The Fos-positive cells were manually counted in at least two sections from each animal and the values tested statistically using the Mann±Whitney U-test and the Wilcoxon-ranked test at the 5% level of signi®cance. The anterior and posterior lobes of the pituitary, but not the intermediate lobe, showed positive NADPH-d reactivity (Fig. 1). The neural (posterior) lobe of capsaicin-treated animals showed a signi®cant upregulation of NADPH-d staining compared with vehicle-treated animals (265 ^ 33 vs. 163 ^ 27 arbitrary units per section; P , 0:01, Mann± Whitney U-test; Fig. 1a,b). Within the anterior lobe, capsaicin-treated rats showed a marked upregulation of NADPH-d compared with vehicle-treated animals (Fig. 1c,d). Capsaicin-treated animals exhibited a relatively stronger NADPH-d staining in the folliculo-stellate cells than was observed in vehicle-treated rats (arrows, Fig. 1c). Compared with d-NAME, pretreatment with l-NAME before capsaicin injection reduced the expression
of NADPH-d in the anterior and posterior lobes (P , 0:01, Wilcoxon test; Fig. 2A,B). Capsaicin activated Fos expression in the intermediate and anterior lobes. Although there was no strict topographical organization of the Fos-positive cells in the anterior lobe, there was a tendency for these cells to be predominantly localized distal to the intermediate lobe. A signi®cantly higher (P , 0:01, Mann±Whitney U-test) activation of Fos expression was observed in the anterior and intermediate lobes after capsaicin injection compared with vehicle (426 ^ 67 vs. 45 ^ 13 cells per section; Fig. 1e,f). The posterior lobe lacked Fos immunoreactive cells. Fos expression in the anterior and intermediate lobes of capsaicintreated animals was signi®cantly attenuated by pretreatment with l-NAME compared with d-NAME (236 ^ 91 vs. 399 ^ 61 cells per section; P , 0:05, Mann±Whitney Utest; Fig. 2C,D). Hence capsaicin-induced activation of cellular response in the anterior and intermediate lobes of the pituitary requires NOS mediation. This observation, however, does not preclude the involvement of other mediator molecules. The results presented in this report favor a regulatory in¯uence of NOS in the effect of capsaicin on the pituitary gland. The major ®nding in this study is capsaicin-induced activation of Fos and NADPH-d expression in the pituitary gland and attenuation of the capsaicin-induced Fos expression by pretreatment with a speci®c NOS inhibitor. The observed reduction in NADPH-d staining in the pituitary gland after l-NAME pretreatment agrees with the ability of NOS inhibitors to block NADPH-d expression. In the neural lobe, this reduction is consistent with the decreased NADPH-d staining in the cell bodies of magnocellular neurons in the hypothalamus [13]. That capsaicin activated marked Fos expression in the intermediate and anterior lobes agrees with previous ®ndings in animals stressed by immobilization [8,17]. Different types of stressors appear to activate the release of adrenocorticotropic hormone (ACTH) from either the intermediate or anterior lobes [15] and predominantly activate Fos expression in ACTH cells [13]. Although there is, as yet, no direct evidence of NOS colocalization with ACTH cells, an increased vascular supply to secretory cells resulting from the perivascular localization of NOS in the anterior lobe [10] and the property of NO as a freely diffusible neurotransmitter potentially allow for a modulation of ACTH [3] and secretion of other hormone(s). Further studies using double labeling techniques will be necessary to determine the detailed pattern of Fos and NADPH-d expression together with other regulators of stress in the anterior lobe of the pituitary gland. The principal neuroendocrine response to acute physical or psychological stress is the synthesis and secretion, by the hypothalamo-pituitary-adrenal axis, of corticotropin-releasing factor (CRF), ACTH and propiomelanocortin-derived peptides as well as corticosteroids [1]. The CRF neurons from the parvocellular paraventricular nucleus of the hypothalamus project to the median eminence where secre-
C.O. Okere et al. / Neuroscience Letters 295 (2000) 73±76
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Fig. 1. Photomicrographs from one set of animals showing NADPH diaphorase reactivity (a±d) and Fos immunoreactive cells (e,f) in the pituitary gland of capsaicin- (a,c,e) and vehicle- (b,d,f) treated animals. The posterior (a vs. b) and anterior (c vs. d, higher magni®cation) lobes of capsaicin-treated animals showed a higher NADPH diaphorase staining. More Fos immunoreactive cells were present in the anterior and intermediate lobes in response to capsaicin (e vs. f). e2 represents a section from regions of the anterior lobe lateral to the intermediate lobe in a coronal plane. Scale bar in (f) 200 mm in (a,b,e,f); 100 mm, in (c,d). Arrows in (c) indicate folliculo-stellate cells. AL, anterior lobe; IL, intermediate lobe; PL, posterior (neural) lobe.
Fig. 2. Photomicrographs from a set of animals showing the effect of the NOS inhibitor-(l-NAME, A,C) or its (control) inactive enantiomer d-NAME (B,D) pretreatment on capsaicin-induced NADPH diaphorase (A,B) and Fos immunoreactivity (C,D) in the pituitary. Pretreatment with l-NAME attenuated the capsaicin-induced Fos expression and NADPH diaphorase in the anterior lobe. Scale bar in (D) 200 mm (A,B) and 400 mm in (C,D).
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tion of CRF into the hypophysial portal blood occurs. In a previous report, capsaicin upregulated the expression of NADPH-d in the internal zone of the median eminence and Fos expression in the arcuate nucleus [11]. The present study showed a capsaicin-induced upregulation of NADPHd in the neural lobe. Since the neural lobe is responsible for the secretion of oxytocin and vasopressin, this suggests that capsaicin may alter the secretion of either or both hormones. Interestingly, chronic capsaicin treatment has been reported to stimulate substance P release in the supraoptic nucleus and to cause antidiuresis as a result of increased release of vasopressin from the neural lobe [18]. The signi®cance of the relatively higher NADPH-d staining in the folliculostellate cells in capsaicin-treated animals observed in this study is obscure. Although NOS expression is upregulated in the hypothalamo-pituitary-adrenal axis during immobilization stress [7] and capsaicin activates immediate early gene expression in the paraventricular nucleus [6], there is no evidence to our knowledge, linking capsaicin-induced cellular activation to an upregulation of NOS activity. Taken together with previous observations of capsaicin-induced activation of NOS neurons in the arcuate nucleus, median eminence and hypothalamus [11,13], the present observations indicate that NO is a principal mediator of capsaicin effect on the hypothalamo-pituitary axis. This hypothesis is supported by the ®nding that in animals pretreated with a NOS inhibitor, the capsaicin-induced activation of Fos expression was signi®cantly attenuated. In summary, evidence reported in this study showed that capsaicin treatment caused cellular (Fos) activation and upregulation of NADPH-diaphorase (NOS) reactivity in the pituitary gland. Pretreatment with a NOS inhibitor attenuated the capsaicin-induced cellular activation. Taken together, the present observations suggest that the effect of acute capsaicin on the pituitary gland requires a signi®cant NO mediation. This work was supported by research grants from the Ministry of Education, Science, Sports and Culture of Japan. C.O. Okere was in receipt of a Japan Society for the Promotion of Science Postdoctoral Fellowship. [1] Axelrod, J. and Reisine, T.D., Stress hormones: their interaction and regulation, Science, 224 (1984) 452±459. [2] Bonaz, B. and TacheÂ, Y., Corticotropin-releasing factor and systemic capsaicin-sensitive afferents are involved in abdominal surgery-induced Fos expression in the paraventricular nucleus of the hypothalamus, Brain Res., 748 (1997) 12±20. [3] Brunetti, L., Preziosi, P., Ragazzoni, E. and Vacca, M., Involvement of nitric oxide in basal and interleukin 1b-induced CRH and ACTH release in vitro, Life Sci., 53 (1993) PL219± PL222. [4] Garthwaite, J., Garthwaite, G., Palmer, R.M.J. and Moncada, S., NMDA receptor activation induces nitric oxide synthesis from arginine in rat brain slices, Eur. J. Pharmacol., 172 (1989) 413±416.
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