431
Journal of Physiology (1991), 443, pp. 431-439 With 3 figures Printed in Great Britain
ACTH RELEASE INDUCED IN RATS BY NORADRENALINE IS MEDIATED BY PROSTAGLANDIN E2
BY TATSUO WATANABE, AKIO MORIMOTO, KEIKO MORIMOTO, TOMOKI NAKAMORI AND NAOTOSHI MURAKAMI From the Department of Physiology, Yamaguchi University School of Medicine, Ube, Yamaguchi 755, Japan
(Received 28 January 1991) SUMMARY
1. We investigated the involvement of prostaglandin E2 in the development of the adrenocorticotrophic hormone (ACTH) response induced by noradrenaline (NA) in rats. 2. Intravenous (i.v.) injection of NA produced dose-dependent increases in the plasma concentration of ACTH and prostaglandin E2. However, pre-treatment with systemic administration of indomethacin, an inhibitor of prostaglandin synthesis, significantly suppressed this increase in plasma ACTH. 3. The i.v. injection of prostaglandin E2 significantly increased the plasma concentration of ACTH in a dose-dependent manner. In contrast, ACTH responses induced by the i.v. injection of prostaglandin E2 were significantly suppressed by systemic pre-treatment with anti-corticotrophin-releasing factor antibody (antiCRF), although the plasma level of ACTH still increased in comparison to the basal level. 4. These results suggest that NA-stimulated prostaglandin release is involved in the ACTH response induced by NA. In addition, it is likely that CRF may be responsible for a portion of the ACTH response induced by i.v. injection of prostaglandin E2. INTRODUCTION
Acute stress has been shown to induce a variety of hormonal responses, including significant increases in the plasma level of catecholamines and adrenocorticotrophic hormone (ACTH). However, still little is known about the trigger(s) that stimulates the release of ACTH during stressful conditions. Recent reports suggest the possibility that the ACTH response during physical or psychological stress is mediated by prostaglandins. Pre-treatment with systemic administration of indomethacin, an inhibitor of prostaglandin synthesis, significantly suppresses the increase in the plasma ACTH induced by swimming (Watanabe, Morimoto, Sakata, Long & Murakami, 1991) or cage-switch stress (Morimoto, Watanabe, Morimoto, Nakamori & Murakami, 1991). In addition, we reported that the intrahypothalamic injection of prostaglandin E2 produced a significant increase in the plasma concentration of ACTH (Morimoto, Murakami, Nakamori, Sakata & Watanabe, MS 9107
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T. WATANABE AND OTHERS
1989; Watanabe, Morimoto, Sakata & Murakami, 1990) and that this ACTH response was completely suppressed by pre-treatment with the intravenous injection of anti-corticotrophin-releasing factor antibody (Watanabe et al. 1990). Therefore, we suggested that prostaglandin E2 induces the ACTH response by acting on the hypothalamus to stimulate corticotrophin-releasing factor (CRF) secretion into the hypophyseal portal vein. CRF subsequently stimulates the pituitary cells to secrete ACTH. At the present time, however, the mechanism by which stress stimulates prostaglandin synthesis is not known. Animals generally respond to stress in a stereotyped manner that includes the activation of the sympathetic nervous system. Sympathetic nerve terminals release noradrenaline (NA), and consequently plasma levels of catecholamines increase. It has been reported that the plasma concentrations of ACTH increase after intravenous injection of catecholamine (Berkenbosch, Vermes, Binnekade & Tilders, 1981; Tilders, Berkenbosch & Smelik, 1982; Hary, Dupouy & Chatelain, 1984). In addition, previous studies have shown that NA stimulates the release of prostaglandin E2 in peripheral tissues (Hedqvist, 1977) and the central nervous system (Busija & Leffler, 1987). Therefore we speculated that during stressful conditions, NA release into the circulation stimulates the prostaglandin synthesis and, consequently, the ACTH response is induced. The present study was carried out to investigate the role of prostaglandins in the NA-induced ACTH response in rats. We found that intravenous injections of NA increased the plasma concentrations of ACTH and prostaglandin E2. In addition, the pre-treatment with systemic administration of indomethacin significantly suppressed the increase in the plasma ACTH induced by intravenous injection of NA. These results suggest that prostaglandins are involved in the NA-induced ACTH response. METHODS
Male albino rats (Wistar strain) weighing 280-360 g were used in this study. The rats were housed in individual cages in a room maintained at 26±1 °C, a temperature within the thermoneutral zone for rats, with a 12-12 h light-dark cycle, with light on 07.00 h. Tap water and rodent chow were provided ad libitum. This study consisted of four experiments. In Expt 1, rats were intravenously injected with NA or saline. In Expt 2, rats were intravenously administered indomethacin (INDO) or a control vehicle 15 min before intravenous injection of NA. In Expt 3, rats were intravenously injected with prostaglandin E2 or saline. In Expt 4, rats were intravenously administered anti-CRF antibody (anti-CRF) or normal rabbit serum (NRS) as a control 15 min before intravenous injection of prostaglandin E2. For blood sampling and intravenous injections, the rats were implanted with intravenous catheters. While the rats were under general anaesthesia (pentobarbitone, 50 mg/kg, i.P.), polyvinyl tubing was inserted from the jugular vein such that the tip of the tubing was located in the superior caval vein (SCV) near the right atrium. The free end of the catheter was passed subcutaneously to the mid-scapular region, where it was exteriorized through the skin on the dorsal side of the neck. The patency of the catheter was maintained by daily flushes with heparinized 0 9 % saline (50 U/ml). In addition, each rat was handled for 10 min every day for at least 5 days prior to the start of the experiments to accustom the animals to the experimenters. For i.v. injection, NA-HCl was dissolved in sterile saline at a concentration of 0 1 or 0 01 mg/ml. Prostaglandin E2 was dissolved in sterile saline containing 0 5 % ethanol at a concentration of 1 or 0 1 mg/ml. We used saline containing 0'5 % ethanol as the control vehicle for prostaglandin E2 solution. Indomethacin was dissolved in sterile saline containing 4 % sodium bicarbonate at a concentration of 1 mg/ml. We used saline containing 4 % sodium bicarbonate as the control vehicle for indomethacin. Anti-corticotrophin-releasing factor antibody (anti-CRF, Cambridge Research
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Biomedicals) was also dissolved in sterile saline. Injection doses in each experimental group, including appropriate control injections, are described in the Results. On the day of the experiments, rats were housed in individual cages and were allowed to move freely, with access to food and water ad libitum. The experiments were carried out at an ambient temperature of 26 +1 'C. i.v. injections of NA, prostaglandin E2 or saline via the SCV cannula were performed at about 12-00 h. To measure the plasma concentration of ACTH, about 0 5 ml of blood was withdrawn through the cannulae. The blood samples were taken from each rat three times: 30 min before and 20 and 90 min after intravenous injection of NA or prostaglandin E2. Blood was collected into test-tubes containing EDTA (1 mg/ml blood) to measure the ACTH concentration. To measure the plasma concentration of prostaglandin E2, about 1P0 ml of blood was withdrawn. Blood samples were taken twice from each rat, 30 min before and 20 min after intravenous injection of NA and collected into test-tubes containing EDTA (1 mg/ml blood) and indomethacin (0 5 mg/ml blood). The blood was centrifuged at 2000 r.p.m. for 15 min at 4 'C. The plasma was then transferred to new test-tubes and stored at -40 'C until further analysis. The blood cells were resuspended in sterile saline and stored at 4 'C, and returned to each animal after completion of each day of experiments. To determine the ACTH and prostaglandin E2 concentrations, radioimmunoassays were performed, using commercial radioimmunoassay kits (Diagnostic Product Corporation, USA; Amersham, UK). The ACTH or prostaglandin E2 antiserum in the assays exhibited an extremely low cross-reactivity (< 0 5 % for ACTH and < 5 % for prostaglandin E2 antibody) to other compounds. The data were analysed for statistical significance by Student's t test or ANOVA. RESULTS
Figure 1A shows the mean change in the plasma concentration of ACTH after intravenous injections of NA (041 or 0-01 mg/kg), or saline. Statistical comparisons were made between the NA-injected group and the saline injected group. As shown in Fig. 1A, the plasma concentration of ACTH increased significantly 20 min after the injection of a low dose of NA and 20 and 90 min after the injection of a high dose of NA. The increased level of ACTH induced by a high dose of NA was significantly (P < 0 01) greater than that induced by a low dose 20 min after injection. Intravenous injection of saline had no effect on plasma concentration of ACTH after injection. Figure lB shows the effect of systemic pre-treatment with indomethacin (INDO, 1 mg/kg) on the ACTH response induced by intravenous injection of NA (0 1 mg/kg) or saline. INDO or vehicle was intravenously injected 15 min before the injection of NA or saline. Note that the increase in plasma ACTH induced by intravenous injection of NA was significantly suppressed by the pre-treatment with INDO, in comparison to the rats that were injected with vehicle. However, the pre-treatment with INDO 15 min prior to saline injection had no effect on plasma levels of ACTH. Figure 2 shows the mean changes in the plasma concentration of prostaglandin E2 after the intravenous injection of NA (041 and 0 01 mg/kg), or saline. As shown in Fig. 2, the intravenous injection of NA induced significant increases in the plasma concentration of prostaglandin E2 in a dose-dependent manner. No changes in plasma levels of prostaglandin E2 were observed after intravenous injection of saline. Figure 3A shows the mean change in the plasma concentration of ACTH after intravenous injections of prostaglandin E2 (10 and 01 mg/kg) or saline. Prostaglandin E2 produced dose-dependent rises in the plasma concentration of ACTH 20 min after injection, as compared to the rats who received saline. The plasma concentration of ACTH was still significantly elevated 90 min after the
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Journal of Physiology (1991), 443, pp. 431-439 With 3 figures Printed in Great Britain
ACTH RELEASE INDUCED IN RATS BY NORADRENALINE IS MEDIATED BY PROSTAGLANDIN E2
BY TATSUO WATANABE, AKIO MORIMOTO, KEIKO MORIMOTO, TOMOKI NAKAMORI AND NAOTOSHI MURAKAMI From the Department of Physiology, Yamaguchi University School of Medicine, Ube, Yamaguchi 755, Japan
(Received 28 January 1991) SUMMARY
1. We investigated the involvement of prostaglandin E2 in the development of the adrenocorticotrophic hormone (ACTH) response induced by noradrenaline (NA) in rats. 2. Intravenous (i.v.) injection of NA produced dose-dependent increases in the plasma concentration of ACTH and prostaglandin E2. However, pre-treatment with systemic administration of indomethacin, an inhibitor of prostaglandin synthesis, significantly suppressed this increase in plasma ACTH. 3. The i.v. injection of prostaglandin E2 significantly increased the plasma concentration of ACTH in a dose-dependent manner. In contrast, ACTH responses induced by the i.v. injection of prostaglandin E2 were significantly suppressed by systemic pre-treatment with anti-corticotrophin-releasing factor antibody (antiCRF), although the plasma level of ACTH still increased in comparison to the basal level. 4. These results suggest that NA-stimulated prostaglandin release is involved in the ACTH response induced by NA. In addition, it is likely that CRF may be responsible for a portion of the ACTH response induced by i.v. injection of prostaglandin E2. INTRODUCTION
Acute stress has been shown to induce a variety of hormonal responses, including significant increases in the plasma level of catecholamines and adrenocorticotrophic hormone (ACTH). However, still little is known about the trigger(s) that stimulates the release of ACTH during stressful conditions. Recent reports suggest the possibility that the ACTH response during physical or psychological stress is mediated by prostaglandins. Pre-treatment with systemic administration of indomethacin, an inhibitor of prostaglandin synthesis, significantly suppresses the increase in the plasma ACTH induced by swimming (Watanabe, Morimoto, Sakata, Long & Murakami, 1991) or cage-switch stress (Morimoto, Watanabe, Morimoto, Nakamori & Murakami, 1991). In addition, we reported that the intrahypothalamic injection of prostaglandin E2 produced a significant increase in the plasma concentration of ACTH (Morimoto, Murakami, Nakamori, Sakata & Watanabe, MS 9107
432
T. WATANABE AND OTHERS
1989; Watanabe, Morimoto, Sakata & Murakami, 1990) and that this ACTH response was completely suppressed by pre-treatment with the intravenous injection of anti-corticotrophin-releasing factor antibody (Watanabe et al. 1990). Therefore, we suggested that prostaglandin E2 induces the ACTH response by acting on the hypothalamus to stimulate corticotrophin-releasing factor (CRF) secretion into the hypophyseal portal vein. CRF subsequently stimulates the pituitary cells to secrete ACTH. At the present time, however, the mechanism by which stress stimulates prostaglandin synthesis is not known. Animals generally respond to stress in a stereotyped manner that includes the activation of the sympathetic nervous system. Sympathetic nerve terminals release noradrenaline (NA), and consequently plasma levels of catecholamines increase. It has been reported that the plasma concentrations of ACTH increase after intravenous injection of catecholamine (Berkenbosch, Vermes, Binnekade & Tilders, 1981; Tilders, Berkenbosch & Smelik, 1982; Hary, Dupouy & Chatelain, 1984). In addition, previous studies have shown that NA stimulates the release of prostaglandin E2 in peripheral tissues (Hedqvist, 1977) and the central nervous system (Busija & Leffler, 1987). Therefore we speculated that during stressful conditions, NA release into the circulation stimulates the prostaglandin synthesis and, consequently, the ACTH response is induced. The present study was carried out to investigate the role of prostaglandins in the NA-induced ACTH response in rats. We found that intravenous injections of NA increased the plasma concentrations of ACTH and prostaglandin E2. In addition, the pre-treatment with systemic administration of indomethacin significantly suppressed the increase in the plasma ACTH induced by intravenous injection of NA. These results suggest that prostaglandins are involved in the NA-induced ACTH response. METHODS
Male albino rats (Wistar strain) weighing 280-360 g were used in this study. The rats were housed in individual cages in a room maintained at 26±1 °C, a temperature within the thermoneutral zone for rats, with a 12-12 h light-dark cycle, with light on 07.00 h. Tap water and rodent chow were provided ad libitum. This study consisted of four experiments. In Expt 1, rats were intravenously injected with NA or saline. In Expt 2, rats were intravenously administered indomethacin (INDO) or a control vehicle 15 min before intravenous injection of NA. In Expt 3, rats were intravenously injected with prostaglandin E2 or saline. In Expt 4, rats were intravenously administered anti-CRF antibody (anti-CRF) or normal rabbit serum (NRS) as a control 15 min before intravenous injection of prostaglandin E2. For blood sampling and intravenous injections, the rats were implanted with intravenous catheters. While the rats were under general anaesthesia (pentobarbitone, 50 mg/kg, i.P.), polyvinyl tubing was inserted from the jugular vein such that the tip of the tubing was located in the superior caval vein (SCV) near the right atrium. The free end of the catheter was passed subcutaneously to the mid-scapular region, where it was exteriorized through the skin on the dorsal side of the neck. The patency of the catheter was maintained by daily flushes with heparinized 0 9 % saline (50 U/ml). In addition, each rat was handled for 10 min every day for at least 5 days prior to the start of the experiments to accustom the animals to the experimenters. For i.v. injection, NA-HCl was dissolved in sterile saline at a concentration of 0 1 or 0 01 mg/ml. Prostaglandin E2 was dissolved in sterile saline containing 0 5 % ethanol at a concentration of 1 or 0 1 mg/ml. We used saline containing 0'5 % ethanol as the control vehicle for prostaglandin E2 solution. Indomethacin was dissolved in sterile saline containing 4 % sodium bicarbonate at a concentration of 1 mg/ml. We used saline containing 4 % sodium bicarbonate as the control vehicle for indomethacin. Anti-corticotrophin-releasing factor antibody (anti-CRF, Cambridge Research
ACTH AND NORADRENALINE
433
Biomedicals) was also dissolved in sterile saline. Injection doses in each experimental group, including appropriate control injections, are described in the Results. On the day of the experiments, rats were housed in individual cages and were allowed to move freely, with access to food and water ad libitum. The experiments were carried out at an ambient temperature of 26 +1 'C. i.v. injections of NA, prostaglandin E2 or saline via the SCV cannula were performed at about 12-00 h. To measure the plasma concentration of ACTH, about 0 5 ml of blood was withdrawn through the cannulae. The blood samples were taken from each rat three times: 30 min before and 20 and 90 min after intravenous injection of NA or prostaglandin E2. Blood was collected into test-tubes containing EDTA (1 mg/ml blood) to measure the ACTH concentration. To measure the plasma concentration of prostaglandin E2, about 1P0 ml of blood was withdrawn. Blood samples were taken twice from each rat, 30 min before and 20 min after intravenous injection of NA and collected into test-tubes containing EDTA (1 mg/ml blood) and indomethacin (0 5 mg/ml blood). The blood was centrifuged at 2000 r.p.m. for 15 min at 4 'C. The plasma was then transferred to new test-tubes and stored at -40 'C until further analysis. The blood cells were resuspended in sterile saline and stored at 4 'C, and returned to each animal after completion of each day of experiments. To determine the ACTH and prostaglandin E2 concentrations, radioimmunoassays were performed, using commercial radioimmunoassay kits (Diagnostic Product Corporation, USA; Amersham, UK). The ACTH or prostaglandin E2 antiserum in the assays exhibited an extremely low cross-reactivity (< 0 5 % for ACTH and < 5 % for prostaglandin E2 antibody) to other compounds. The data were analysed for statistical significance by Student's t test or ANOVA. RESULTS
Figure 1A shows the mean change in the plasma concentration of ACTH after intravenous injections of NA (041 or 0-01 mg/kg), or saline. Statistical comparisons were made between the NA-injected group and the saline injected group. As shown in Fig. 1A, the plasma concentration of ACTH increased significantly 20 min after the injection of a low dose of NA and 20 and 90 min after the injection of a high dose of NA. The increased level of ACTH induced by a high dose of NA was significantly (P < 0 01) greater than that induced by a low dose 20 min after injection. Intravenous injection of saline had no effect on plasma concentration of ACTH after injection. Figure lB shows the effect of systemic pre-treatment with indomethacin (INDO, 1 mg/kg) on the ACTH response induced by intravenous injection of NA (0 1 mg/kg) or saline. INDO or vehicle was intravenously injected 15 min before the injection of NA or saline. Note that the increase in plasma ACTH induced by intravenous injection of NA was significantly suppressed by the pre-treatment with INDO, in comparison to the rats that were injected with vehicle. However, the pre-treatment with INDO 15 min prior to saline injection had no effect on plasma levels of ACTH. Figure 2 shows the mean changes in the plasma concentration of prostaglandin E2 after the intravenous injection of NA (041 and 0 01 mg/kg), or saline. As shown in Fig. 2, the intravenous injection of NA induced significant increases in the plasma concentration of prostaglandin E2 in a dose-dependent manner. No changes in plasma levels of prostaglandin E2 were observed after intravenous injection of saline. Figure 3A shows the mean change in the plasma concentration of ACTH after intravenous injections of prostaglandin E2 (10 and 01 mg/kg) or saline. Prostaglandin E2 produced dose-dependent rises in the plasma concentration of ACTH 20 min after injection, as compared to the rats who received saline. The plasma concentration of ACTH was still significantly elevated 90 min after the
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