Atrial natriuretic peptide is involved in the ACTH response to stress and glucocorticoid negative feedback in the rat G. Fink, R. C. Dow, D. S. Carroll and H. Dick
Casley,
C. I. Johnston, J.
Bennie,
MRC Brain Metabolism Unit, University Department of Pharmacology, 1 George Edinburgh eh8 9JZ, U.K. *Department of Medicine, Austin Hospital, Heidelberg, Victoria 3084, Australia received
Square,
10 March 1992
ABSTRACT
The role of atrial natriuretic peptide (ANP) in the ACTH response to stress and the glucocorticoid negative feedback control of ACTH release was investigated in adult male homozygous Brattleboro and adrenalectomized Wistar rats respectively, using the technique of immunoneutralization. The relatively low ACTH response to stress and the lack of arginine vasopressin make the homozygous Brattleboro rat a more rigorous and simpler preparation in which to test the hypothesis that ANP is involved in the ACTH response to stress. In both sets of experiments, blood sampling and injection of sheep anti-ANP or control serum were carried out in conscious animals through intra-atrial cannulae implanted 2 days previously under halothane anaesthesia. A 30-s exposure to ether resulted in a brisk twofold increase in the plasma ACTH concentrations in homozygous Brattleboro
INTRODUCTION
Adrenocorticotrophin (ACTH) was generally thought to be under stimulatory control of the central nervous system mediated mainly by corticotrophin releasing factor-41 (CRF-41) and arginine vasopres¬ sin (AVP) (Antoni, 1986; Rivier & Plotsky, 1986; Fink, Robinson & Tannahill, 1988; Sheward & Fink, 1991; Tannahill, Sheward, Robinson & Fink, 1991). However, the effects of cerebral decortication in the
dog (Egdahl, 1961, 1962), hypothalamic deafferentation in the rat (Halász, Vernikos-Danellis & Gorski, 1967) and pituitary isolation in sheep (Engler, Pham, Fullerton et al. 1988) suggested that the central ner¬ vous
system (CNS) may also inhibit ACTH release.
showed that immunoneutralization of Recently atrial natriuretic peptide (ANP) with a highly specific we
rats infused with anti-ANP, but not control serum. The injection of either dexamethasone, a potent glucocorticoid receptor agonist, or corticosterone resulted in a rapid and marked reduction in the plasma concentrations of ACTH in Wistar rats which had been adrenalectomized, under halothane anaesthesia, at least 21 days before experimentation. The inhibitory action of dexamethasone, but not corticosterone, was significantly reduced in animals infused with anti\x=req-\ ANP serum. These results show that the inhibition of ANP release into hypophysial portal blood is probably important for triggering the ACTH response to stress and that ANP may play a role in corticosteroid negative feedback control of ACTH release mediated by type II (glucocorticoid) receptors. Journal of Endocrinology (1992) 135, 37\p=n-\43
sheep anti-ANP serum resulted in an immediate, five¬ fold increase in peripheral plasma ACTH concentra¬ tions which was accompanied by an increase in the plasma concentration of corticosterone (Fink, Dow, Casley et al. 1991). This suggested that ANP is a (and possibly the) physiological ACTH-inhibiting factor, an inference supported by (i) the existence of ANP projections from the paraventricular nuclei to the median eminence (Palkovits, Eskay & Antoni, 1987), (ii) the fact that ANP concentrations are about four times higher in hypophysial portal compared with peripheral blood (Lim, Sheward, Copolov et al. 1990; Sheward, Lim, Alder et al. 1991), and (iii) the fact that ANP can inhibit ACTH release in vitro (Shibasaki, Naruse, Yamauchi et al. 1986; Dayanithi & Antoni, 1990). Our in-vivo immuno-
neutralization studies were confirmed by an indepen¬ dent study in which rabbit anti-ANP sera were used (Antoni, Hunter, Lowry et al. 1992). The purpose of the present study was to determine whether ANP plays a role in (i) the ACTH response to stress, and (ii) glucocorticoid negative feedback inhibition of ACTH secretion. The role of ANP was determined by the technique of immunoneutraliza¬ tion. The possible role of ANP in the hypothalamicpituitary ACTH response to stress was studied in homozygous Brattleboro rats for two interrelated reasons. First, the 'basal' plasma concentrations of ACTH and corticosterone and the ACTH and corticosterone responses to a 2-min ether stress are significantly lower in homozygous Brattleboro com¬ pared with Long Evans rats (Buckingham, 1982; Conte-Devolx, Oliver, Giraud et al. 1982). We rea¬ soned, therefore, that if the role of ANP was crucial in the ACTH response to stress, an immunoneutraliz¬ ation study in homozygous Brattelboro rats would provide the rigorous proof for this hypothesis. Secondly, homozygous Brattleboro rats lack AVP and, thereby, one important variable is eliminated, leaving just CRF-41 versus the putative inhibitory
factor, ANP.
MATERIALS AND METHODS
Animals
homozygous Brattleboro rats (bred in the Department or purchased from Biomedicai Services, University of Leeds) and male Wistar rats (purchased from Charles River UK Ltd, Margate, Kent, U.K.)
Adult male
used. The animals were maintained under con¬ trolled lighting (lights on 05.00 h-19.00 h) and tem¬ perature (22 °C) and allowed free access to diet 41 (Oxoid, Basingstoke, Hants, U.K.) and tap water.
were
Antisera for immunoneutralization
The anti-ANP sera used for immunoneutralization were raised in sheep and were SI 17, as used in Fink et al. (1991) and SI 18. The titre of SI 18 was identical to that of SI 17. Possible role of ANP in the stress response The possible role of ANP in mediating the hypothalamic-pituitary response to stress was studied in male homozygous Brattleboro rats with a body weight of 200-300 g. Two days before the stress experiment the animals were anaesthetized with halothane and
implanted with an indwelling intra-atrial cannula (Fink et al. 1991). The stress experiment was carried
out in the conscious animals between 13.00 and
18.30 h. Our previous study (Fink et al. 1991) showed that the infusion of 0-8 ml anti-ANP sheep serum (SI 17) caused a relatively massive increase in plasma ACTH concentrations which could conceivably mask the effects of a mild stress (Fink et al. 1991). In the present experiment, therefore, the dose of sheep antiANP serum was reduced to 0-3 ml rather than 0-8 ml. As before, 0- 3 ml blood samples were withdrawn through the indwelling intra-atrial cannula 30 min before and 15, 30 and 60 min after the i.v. infusion of 0-3 ml of either the sheep anti-ANP or control (nonimmune) sheep serum (supplied by the Scottish Anti¬ body Production Unit, Carluke, Lanarkshire, U.K.). The sera were infused over a period of 30 s. The ani¬ mals were then exposed to ether vapour for precisely 30 s and 0-3 ml blood samples were again withdrawn from the intra-atrial cannula at 15, 30, 60 and 240 min after the stress (75, 90, 120 and 300 min respectively, after serum infusion). An additional con¬ trol group was infused with the anti-ANP serum and treated as above except that no stress was applied. The experiments were designed so that the same number of control and anti-ANP-treated animals were studied on the same day and time and, secondly, that the animals were exposed to ether only after the 'basal' samples had all been taken from each animal. The blood samples were immediately mixed in chilled plastic centrifuge tubes with 1000 kallikrein inactivator units of aprotinin (Trasylol; Bayer UK Ltd, Newbury, Berks, U.K.) and kept on ice. The tubes were centrifuged within a short period of blood sampling and the plasma stored at —40 °C. The role of ANP in ACTH release
glucocorticoid feedback control
of
Male rats were adrenalectomized under halothane anaesthesia and allowed at least 21 days to recover. During this period the animals were allowed free access to water containing 20 g glucose and 9 g NaCl/1. During the first 5 post-operative days the drinking water also contained 0- 8 g chlortetracycline/1. An intra-atrial cannula was implanted 2 days before experimentation. Two sets of experiments were car¬ ried out to determine the role of ANP in the negative feedback of either corticosterone or the glucocorticoid receptor agonist, dexamethasone. Both sets of experi¬ ments were carried out between 13.00 and 18.30 h. Completeness of adrenalectomy was checked at
autopsy. Corticosterone
study A 0- 3 ml blood sample was taken from the conscious animals 30 min before the i.v. infusion of 0-2 or 0-3 ml
sheep anti-ANP serum or control (non-immune) period of 30 s. Corticosterone (Sigma Chemical Company Ltd, Poole, Dorset, U.K.; lot 129F0829) was then injected 5 min after the injection of the anti-ANP or control serum, at a dosage of
80- (a)
serum over a
60-
100 pg i.v. and 200 pg s.c. in volumes of 0-2 and 0-4 ml. The corticosterone was initially dissolved in 100% ethanol and then diluted stepwise with 0-9% (w/v) NaCl to give a final solution of ethanol : saline of 1:39 (v/v). Blood samples were taken 30 and 60 min after corticosterone administration and the injections of corticosterone were repeated. A further two blood samples were taken at 30 and 60 min after the second corticosterone injection and the cortico¬ sterone injection was repeated. A final blood sample was taken 30 min after the last corticosterone
7.
Casley,
C. I. Johnston, J.
Bennie,
MRC Brain Metabolism Unit, University Department of Pharmacology, 1 George Edinburgh eh8 9JZ, U.K. *Department of Medicine, Austin Hospital, Heidelberg, Victoria 3084, Australia received
Square,
10 March 1992
ABSTRACT
The role of atrial natriuretic peptide (ANP) in the ACTH response to stress and the glucocorticoid negative feedback control of ACTH release was investigated in adult male homozygous Brattleboro and adrenalectomized Wistar rats respectively, using the technique of immunoneutralization. The relatively low ACTH response to stress and the lack of arginine vasopressin make the homozygous Brattleboro rat a more rigorous and simpler preparation in which to test the hypothesis that ANP is involved in the ACTH response to stress. In both sets of experiments, blood sampling and injection of sheep anti-ANP or control serum were carried out in conscious animals through intra-atrial cannulae implanted 2 days previously under halothane anaesthesia. A 30-s exposure to ether resulted in a brisk twofold increase in the plasma ACTH concentrations in homozygous Brattleboro
INTRODUCTION
Adrenocorticotrophin (ACTH) was generally thought to be under stimulatory control of the central nervous system mediated mainly by corticotrophin releasing factor-41 (CRF-41) and arginine vasopres¬ sin (AVP) (Antoni, 1986; Rivier & Plotsky, 1986; Fink, Robinson & Tannahill, 1988; Sheward & Fink, 1991; Tannahill, Sheward, Robinson & Fink, 1991). However, the effects of cerebral decortication in the
dog (Egdahl, 1961, 1962), hypothalamic deafferentation in the rat (Halász, Vernikos-Danellis & Gorski, 1967) and pituitary isolation in sheep (Engler, Pham, Fullerton et al. 1988) suggested that the central ner¬ vous
system (CNS) may also inhibit ACTH release.
showed that immunoneutralization of Recently atrial natriuretic peptide (ANP) with a highly specific we
rats infused with anti-ANP, but not control serum. The injection of either dexamethasone, a potent glucocorticoid receptor agonist, or corticosterone resulted in a rapid and marked reduction in the plasma concentrations of ACTH in Wistar rats which had been adrenalectomized, under halothane anaesthesia, at least 21 days before experimentation. The inhibitory action of dexamethasone, but not corticosterone, was significantly reduced in animals infused with anti\x=req-\ ANP serum. These results show that the inhibition of ANP release into hypophysial portal blood is probably important for triggering the ACTH response to stress and that ANP may play a role in corticosteroid negative feedback control of ACTH release mediated by type II (glucocorticoid) receptors. Journal of Endocrinology (1992) 135, 37\p=n-\43
sheep anti-ANP serum resulted in an immediate, five¬ fold increase in peripheral plasma ACTH concentra¬ tions which was accompanied by an increase in the plasma concentration of corticosterone (Fink, Dow, Casley et al. 1991). This suggested that ANP is a (and possibly the) physiological ACTH-inhibiting factor, an inference supported by (i) the existence of ANP projections from the paraventricular nuclei to the median eminence (Palkovits, Eskay & Antoni, 1987), (ii) the fact that ANP concentrations are about four times higher in hypophysial portal compared with peripheral blood (Lim, Sheward, Copolov et al. 1990; Sheward, Lim, Alder et al. 1991), and (iii) the fact that ANP can inhibit ACTH release in vitro (Shibasaki, Naruse, Yamauchi et al. 1986; Dayanithi & Antoni, 1990). Our in-vivo immuno-
neutralization studies were confirmed by an indepen¬ dent study in which rabbit anti-ANP sera were used (Antoni, Hunter, Lowry et al. 1992). The purpose of the present study was to determine whether ANP plays a role in (i) the ACTH response to stress, and (ii) glucocorticoid negative feedback inhibition of ACTH secretion. The role of ANP was determined by the technique of immunoneutraliza¬ tion. The possible role of ANP in the hypothalamicpituitary ACTH response to stress was studied in homozygous Brattleboro rats for two interrelated reasons. First, the 'basal' plasma concentrations of ACTH and corticosterone and the ACTH and corticosterone responses to a 2-min ether stress are significantly lower in homozygous Brattleboro com¬ pared with Long Evans rats (Buckingham, 1982; Conte-Devolx, Oliver, Giraud et al. 1982). We rea¬ soned, therefore, that if the role of ANP was crucial in the ACTH response to stress, an immunoneutraliz¬ ation study in homozygous Brattelboro rats would provide the rigorous proof for this hypothesis. Secondly, homozygous Brattleboro rats lack AVP and, thereby, one important variable is eliminated, leaving just CRF-41 versus the putative inhibitory
factor, ANP.
MATERIALS AND METHODS
Animals
homozygous Brattleboro rats (bred in the Department or purchased from Biomedicai Services, University of Leeds) and male Wistar rats (purchased from Charles River UK Ltd, Margate, Kent, U.K.)
Adult male
used. The animals were maintained under con¬ trolled lighting (lights on 05.00 h-19.00 h) and tem¬ perature (22 °C) and allowed free access to diet 41 (Oxoid, Basingstoke, Hants, U.K.) and tap water.
were
Antisera for immunoneutralization
The anti-ANP sera used for immunoneutralization were raised in sheep and were SI 17, as used in Fink et al. (1991) and SI 18. The titre of SI 18 was identical to that of SI 17. Possible role of ANP in the stress response The possible role of ANP in mediating the hypothalamic-pituitary response to stress was studied in male homozygous Brattleboro rats with a body weight of 200-300 g. Two days before the stress experiment the animals were anaesthetized with halothane and
implanted with an indwelling intra-atrial cannula (Fink et al. 1991). The stress experiment was carried
out in the conscious animals between 13.00 and
18.30 h. Our previous study (Fink et al. 1991) showed that the infusion of 0-8 ml anti-ANP sheep serum (SI 17) caused a relatively massive increase in plasma ACTH concentrations which could conceivably mask the effects of a mild stress (Fink et al. 1991). In the present experiment, therefore, the dose of sheep antiANP serum was reduced to 0-3 ml rather than 0-8 ml. As before, 0- 3 ml blood samples were withdrawn through the indwelling intra-atrial cannula 30 min before and 15, 30 and 60 min after the i.v. infusion of 0-3 ml of either the sheep anti-ANP or control (nonimmune) sheep serum (supplied by the Scottish Anti¬ body Production Unit, Carluke, Lanarkshire, U.K.). The sera were infused over a period of 30 s. The ani¬ mals were then exposed to ether vapour for precisely 30 s and 0-3 ml blood samples were again withdrawn from the intra-atrial cannula at 15, 30, 60 and 240 min after the stress (75, 90, 120 and 300 min respectively, after serum infusion). An additional con¬ trol group was infused with the anti-ANP serum and treated as above except that no stress was applied. The experiments were designed so that the same number of control and anti-ANP-treated animals were studied on the same day and time and, secondly, that the animals were exposed to ether only after the 'basal' samples had all been taken from each animal. The blood samples were immediately mixed in chilled plastic centrifuge tubes with 1000 kallikrein inactivator units of aprotinin (Trasylol; Bayer UK Ltd, Newbury, Berks, U.K.) and kept on ice. The tubes were centrifuged within a short period of blood sampling and the plasma stored at —40 °C. The role of ANP in ACTH release
glucocorticoid feedback control
of
Male rats were adrenalectomized under halothane anaesthesia and allowed at least 21 days to recover. During this period the animals were allowed free access to water containing 20 g glucose and 9 g NaCl/1. During the first 5 post-operative days the drinking water also contained 0- 8 g chlortetracycline/1. An intra-atrial cannula was implanted 2 days before experimentation. Two sets of experiments were car¬ ried out to determine the role of ANP in the negative feedback of either corticosterone or the glucocorticoid receptor agonist, dexamethasone. Both sets of experi¬ ments were carried out between 13.00 and 18.30 h. Completeness of adrenalectomy was checked at
autopsy. Corticosterone
study A 0- 3 ml blood sample was taken from the conscious animals 30 min before the i.v. infusion of 0-2 or 0-3 ml
sheep anti-ANP serum or control (non-immune) period of 30 s. Corticosterone (Sigma Chemical Company Ltd, Poole, Dorset, U.K.; lot 129F0829) was then injected 5 min after the injection of the anti-ANP or control serum, at a dosage of
80- (a)
serum over a
60-
100 pg i.v. and 200 pg s.c. in volumes of 0-2 and 0-4 ml. The corticosterone was initially dissolved in 100% ethanol and then diluted stepwise with 0-9% (w/v) NaCl to give a final solution of ethanol : saline of 1:39 (v/v). Blood samples were taken 30 and 60 min after corticosterone administration and the injections of corticosterone were repeated. A further two blood samples were taken at 30 and 60 min after the second corticosterone injection and the cortico¬ sterone injection was repeated. A final blood sample was taken 30 min after the last corticosterone
7.
