Regulatory Peptides, 41 (1992) 171-181 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-0115/92/$05.00
171
R E G P E P 01224
Effects of atrial natriuretic factor on norepinephrine release in the rat hypothalamus Marcelo S. Vatta, Mariana L. Papouchado, Ana S. Locatelli, Liliana G. Bianciotti and Belisario E. Fern/tndez Catedra de Fisiopatologia and Programa de Sistemas Vasodepresores - Consejo Nacional de Investigaciones Cientificas y Tecnicas (PROSIVAD - CONICET), Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Buenos Aires (Argentina) (Received 24 March 1992; revised version received 19 June 1992; accepted 23 June 1992)
Key words: Catecholamine release; Central nervous system; Calcium free medium; Calcium channel blocker
Summary The effects of atrial natriuretic factor on the mechanisms involved in norepinephrine release were studied 'in vitro' in slices of Wistar rat hypothalamus. Atrial natriuretic factor (10, 50 and 100 nM) decreased spontaneous [3H]norepinephrine secretion in a concentration dependent way. In addition, the peptide (10 nM) also reduced acetylcholine induced output of norepinephrine. The atrial factor (10 nM) was unable to alter the amine secretion when the incubation medium was deprived of calcium or when a calcium channel blocker such as diltiazem (100/~M) was added. In conclusion, atrial natriuretic factor reduced both spontaneous and acetylcholine evoked [3H]norepinephrine release in the rat hypothalamus. These findings suggest that the atrial natriuretic factor may alter catecholamine secretion by modifying the calcium available for the exocytotic process of catecholamine output.
Correspondence to: M.S. Vatta, C/ltedra de Fisiopatologia and Programa de Sistemas Vasodepresores Consejo Nacional de Investigaciones Cientificas y Trcnicas (PROSIVAD - CONICET), Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Junin 956 - 5to piso, 1113 Buenos Aires, Argentina.
172 Introduction
Atrial natriuretic factor (ANF), a newly discovered hormone, is produced by the mammalian cardiac atria and stored in specific granules that vary according to salt and water intake [ 1-3]. Several lines of evidence suggest that ANF is involved in the control of water and electrolyte balance as well as blood pressure regulation [4-6]. In addition, the atrial factor decreases the peripheral vascular resistance through vasodilatation and reduces blood circulation volume through natriuretic and diuretic mechanisms [ 1,3,7-9]. Moreover, ANF antagonizes angiotensin II (ANG II) peripheral and central effects such as constriction of vascular smooth muscle [ 10], glomerular and tubular renal processes [ 11 ], aldosterone secretion [ 12] and vasopressin release [ 13]. Several ANG lI central actions as induced drinking and salt apetite, are also antagonized by ANF [14]. All these actions prove that ANF is a physiological antagonist of ANG II. We have previously reported that ANF modulates sympathetic neurotransmission, since it increases norepinephrine (NE) uptake in the central nervous system being this effect antagonized by ANG II and III[ 15-18]. The existence of ANF binding sites have been described in several regions of the central nervous system, such as hypothalamus, closely related to cardiocirculatory centers. In addition, ANF and ANG II receptors are located in the same areas of the brain [ 19-21]. Therefore, we considered it of interest to study the role of ANF in the regulation of neuronal NE release in the rat hypothalamus provided that this region is involved in the regulation of blood arterial pressure and neuroendocrine processes and besides in this region NE endogenous levels are considerably high [22-24]. We investigated ANF effects on: (a) spontaneous neuronal NE release; (b) NE release evoked by acetylcholine (Ach); (c) NE output in a calcium free medium (CFM) and (d) NE secretion in the presence of a calcium channel blocker, diltiazem (DTZ).
Materials and Methods
Animals Male Wistar rats weighing between 200-250 g were used in the experiments. The animals were housed in steel cages and maintained at a temperature of 22-24°C in a controlled room with 12 h light/dark cycle (light from 7:00 to 19:00 h). All animals were given free access to water and food. Drugs and incubation solutions The following drugs were used in the experiments: d/l (3H) HC1 NE (New England Nuclear, Boston, MA, USA) of 0.32 #Ci//~g of specific activity; rat ANF (99-126) (Peninsula Lab., Belmont, CA, USA); Ach, eserine (Es) and EGTA (Sigma, St. Louis, USA); DTZ (Roemmers Lab., Buenos Aires, Argentina). Incubation solutions: (a) standard Krebs bicarbonate solution of the following composition (mM): NaCI, 118; KCI, 4.7; MgCI2, 1.2; NaH2PO4, 1.0; CaC12, 2.5; EDTANa, 0.004; dextrose, 11.1; NaHCO 3, 25.0; ascorbic acid, 0.11; (b) CFM solution: this
173 was similar to the standard Krebs bicarbonate solution except for CaC12 which was replaced by an osmotically equivalent amount of sucrose and 0.1 mM E G T A that was added to the medium. Protocols
Animals were decapitated between 10:00 and 12:00 a.m. to avoid circadian variations [25]. Brains were quickly removed, hypothalami were immediately dissected, cooled and weighed. Slices of about 1 mm were cut and then transfered into a glass tube which had a mesh of nylon fitted at the bottom to allow free interchange with the medium. The slices were pre-incubated in a Dubnoff incubator for 15 min at 37 °C (pH 7.4) and bubbled with carbogen (95 ~o 02 and 5 ~o CO2) under continuous shaking with 2 ml of standard Krebs solution. N E stores were labelled during the incubation period (30 rain) with 2.5/~Ci/ml of [3H]NE. The tissues were then washed for 90 min with a standard Krebs solution (in three consecutive 30 min washing periods), to avoid extracellular release and extraneuronal uptake of NE. The tissues were then incubated for 24 min and eight consecutive samples of the incubation medium were collected every 3 min. The first three samples belonged to the basal release period. The second three samples corresponded to the experimental periods and the last two samples corresponded to the post-experimental periods. In each of the three experimental periods ANF, other drugs and the incubation solutions were added in order to test their effects of NE output: - Effects of ANF on spontaneous [3H]NE release: (a)control group (incubated only with standard Krebs solution) and (b), (c) and (d) incubated with 10, 50 and 100 nM ANF, respectively. - Effects of A N F on [3H]NE secretion evoked by Ach: (a) control group; (b) incubated with 100/~M Ach/10 #M Es and (c) incubated with 100 # M Ach/10 #M Es + 10 nM ANF. Effects of ANF on [3H]NE output in a CFM: (a) control group; (b) incubated with a CFM and (c) incubated with a CFM and 10 nM ANF. - Effects of A N F on [3H]NE secretion in a CFM + Ach/Es: (a) incubated with a CFM; (b) incubated with C F M + 100 /tM Ach/10 #M Es and (c) incubated with C F M + 100 # M Ach/10/~M Es + 10 nM ANF. Effects of ANF on [3H]NE release in the presence of a calcium channel blocker: (a) control group; (b) incubated with 100 # M DTZ and (c) incubated with 100/~M DTZ + 10 nM ANF. - Effects of ANF on [3H]NE output evoked by Ach/Es in presence of a calcium channel blocker: (a) incubated with 100/~M Ach/10/~M Es; (b) incubated with 100 /~M Ach/10 /~M Es + 100 # M DTZ and (c) incubated with 100 # M Ach/10 #M Es + 100/~M DTZ + 10 nM ANF. The [3H ]NE activity of all samples was meassured in a Packard scintillation counter (model 240CL/D) by usual scintillation counting methods. It is assumed that the material counted corresponds to [ 3H ] NE. In addition, recent investigations have shown that A N F decreases MAO activity [26]. -
-
174 • :Control • :lOnM 1.1
Iii Z
A N F (7)
u : 5 O H M A N F (5)
i
:]: G~
(7)
o : 1OOnM A N F (5)
lo
"3 q~ O
0.8
~U
0.7 tO
0.6
O
0.5
h
B
I
I
I
I
I
E1
E2
E3
P1
P2
Fig. 1. Effects of A N F on spontaneous [ 3 H ] N E release. Number o f determinations are given in parenthesis. * P < 0.05 when compared to control.
3.C
.
•
:Control
/
•
:IOOuM
/
*
(6) Ach/IOuM
o :IOOuM Ach/IOuM
E S (7)
ES +
•
LL
I
B
El1
I
E2
I
E3
p
~
I
P2
Fig. 2. Effects of A N F on evoked [3H]NE release with Ach. Number of determinations are shown in parenthesis. • P
171
R E G P E P 01224
Effects of atrial natriuretic factor on norepinephrine release in the rat hypothalamus Marcelo S. Vatta, Mariana L. Papouchado, Ana S. Locatelli, Liliana G. Bianciotti and Belisario E. Fern/tndez Catedra de Fisiopatologia and Programa de Sistemas Vasodepresores - Consejo Nacional de Investigaciones Cientificas y Tecnicas (PROSIVAD - CONICET), Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Buenos Aires (Argentina) (Received 24 March 1992; revised version received 19 June 1992; accepted 23 June 1992)
Key words: Catecholamine release; Central nervous system; Calcium free medium; Calcium channel blocker
Summary The effects of atrial natriuretic factor on the mechanisms involved in norepinephrine release were studied 'in vitro' in slices of Wistar rat hypothalamus. Atrial natriuretic factor (10, 50 and 100 nM) decreased spontaneous [3H]norepinephrine secretion in a concentration dependent way. In addition, the peptide (10 nM) also reduced acetylcholine induced output of norepinephrine. The atrial factor (10 nM) was unable to alter the amine secretion when the incubation medium was deprived of calcium or when a calcium channel blocker such as diltiazem (100/~M) was added. In conclusion, atrial natriuretic factor reduced both spontaneous and acetylcholine evoked [3H]norepinephrine release in the rat hypothalamus. These findings suggest that the atrial natriuretic factor may alter catecholamine secretion by modifying the calcium available for the exocytotic process of catecholamine output.
Correspondence to: M.S. Vatta, C/ltedra de Fisiopatologia and Programa de Sistemas Vasodepresores Consejo Nacional de Investigaciones Cientificas y Trcnicas (PROSIVAD - CONICET), Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Junin 956 - 5to piso, 1113 Buenos Aires, Argentina.
172 Introduction
Atrial natriuretic factor (ANF), a newly discovered hormone, is produced by the mammalian cardiac atria and stored in specific granules that vary according to salt and water intake [ 1-3]. Several lines of evidence suggest that ANF is involved in the control of water and electrolyte balance as well as blood pressure regulation [4-6]. In addition, the atrial factor decreases the peripheral vascular resistance through vasodilatation and reduces blood circulation volume through natriuretic and diuretic mechanisms [ 1,3,7-9]. Moreover, ANF antagonizes angiotensin II (ANG II) peripheral and central effects such as constriction of vascular smooth muscle [ 10], glomerular and tubular renal processes [ 11 ], aldosterone secretion [ 12] and vasopressin release [ 13]. Several ANG lI central actions as induced drinking and salt apetite, are also antagonized by ANF [14]. All these actions prove that ANF is a physiological antagonist of ANG II. We have previously reported that ANF modulates sympathetic neurotransmission, since it increases norepinephrine (NE) uptake in the central nervous system being this effect antagonized by ANG II and III[ 15-18]. The existence of ANF binding sites have been described in several regions of the central nervous system, such as hypothalamus, closely related to cardiocirculatory centers. In addition, ANF and ANG II receptors are located in the same areas of the brain [ 19-21]. Therefore, we considered it of interest to study the role of ANF in the regulation of neuronal NE release in the rat hypothalamus provided that this region is involved in the regulation of blood arterial pressure and neuroendocrine processes and besides in this region NE endogenous levels are considerably high [22-24]. We investigated ANF effects on: (a) spontaneous neuronal NE release; (b) NE release evoked by acetylcholine (Ach); (c) NE output in a calcium free medium (CFM) and (d) NE secretion in the presence of a calcium channel blocker, diltiazem (DTZ).
Materials and Methods
Animals Male Wistar rats weighing between 200-250 g were used in the experiments. The animals were housed in steel cages and maintained at a temperature of 22-24°C in a controlled room with 12 h light/dark cycle (light from 7:00 to 19:00 h). All animals were given free access to water and food. Drugs and incubation solutions The following drugs were used in the experiments: d/l (3H) HC1 NE (New England Nuclear, Boston, MA, USA) of 0.32 #Ci//~g of specific activity; rat ANF (99-126) (Peninsula Lab., Belmont, CA, USA); Ach, eserine (Es) and EGTA (Sigma, St. Louis, USA); DTZ (Roemmers Lab., Buenos Aires, Argentina). Incubation solutions: (a) standard Krebs bicarbonate solution of the following composition (mM): NaCI, 118; KCI, 4.7; MgCI2, 1.2; NaH2PO4, 1.0; CaC12, 2.5; EDTANa, 0.004; dextrose, 11.1; NaHCO 3, 25.0; ascorbic acid, 0.11; (b) CFM solution: this
173 was similar to the standard Krebs bicarbonate solution except for CaC12 which was replaced by an osmotically equivalent amount of sucrose and 0.1 mM E G T A that was added to the medium. Protocols
Animals were decapitated between 10:00 and 12:00 a.m. to avoid circadian variations [25]. Brains were quickly removed, hypothalami were immediately dissected, cooled and weighed. Slices of about 1 mm were cut and then transfered into a glass tube which had a mesh of nylon fitted at the bottom to allow free interchange with the medium. The slices were pre-incubated in a Dubnoff incubator for 15 min at 37 °C (pH 7.4) and bubbled with carbogen (95 ~o 02 and 5 ~o CO2) under continuous shaking with 2 ml of standard Krebs solution. N E stores were labelled during the incubation period (30 rain) with 2.5/~Ci/ml of [3H]NE. The tissues were then washed for 90 min with a standard Krebs solution (in three consecutive 30 min washing periods), to avoid extracellular release and extraneuronal uptake of NE. The tissues were then incubated for 24 min and eight consecutive samples of the incubation medium were collected every 3 min. The first three samples belonged to the basal release period. The second three samples corresponded to the experimental periods and the last two samples corresponded to the post-experimental periods. In each of the three experimental periods ANF, other drugs and the incubation solutions were added in order to test their effects of NE output: - Effects of ANF on spontaneous [3H]NE release: (a)control group (incubated only with standard Krebs solution) and (b), (c) and (d) incubated with 10, 50 and 100 nM ANF, respectively. - Effects of A N F on [3H]NE secretion evoked by Ach: (a) control group; (b) incubated with 100/~M Ach/10 #M Es and (c) incubated with 100 # M Ach/10 #M Es + 10 nM ANF. Effects of ANF on [3H]NE output in a CFM: (a) control group; (b) incubated with a CFM and (c) incubated with a CFM and 10 nM ANF. - Effects of A N F on [3H]NE secretion in a CFM + Ach/Es: (a) incubated with a CFM; (b) incubated with C F M + 100 /tM Ach/10 #M Es and (c) incubated with C F M + 100 # M Ach/10/~M Es + 10 nM ANF. Effects of ANF on [3H]NE release in the presence of a calcium channel blocker: (a) control group; (b) incubated with 100 # M DTZ and (c) incubated with 100/~M DTZ + 10 nM ANF. - Effects of ANF on [3H]NE output evoked by Ach/Es in presence of a calcium channel blocker: (a) incubated with 100/~M Ach/10/~M Es; (b) incubated with 100 /~M Ach/10 /~M Es + 100 # M DTZ and (c) incubated with 100 # M Ach/10 #M Es + 100/~M DTZ + 10 nM ANF. The [3H ]NE activity of all samples was meassured in a Packard scintillation counter (model 240CL/D) by usual scintillation counting methods. It is assumed that the material counted corresponds to [ 3H ] NE. In addition, recent investigations have shown that A N F decreases MAO activity [26]. -
-
174 • :Control • :lOnM 1.1
Iii Z
A N F (7)
u : 5 O H M A N F (5)
i
:]: G~
(7)
o : 1OOnM A N F (5)
lo
"3 q~ O
0.8
~U
0.7 tO
0.6
O
0.5
h
B
I
I
I
I
I
E1
E2
E3
P1
P2
Fig. 1. Effects of A N F on spontaneous [ 3 H ] N E release. Number o f determinations are given in parenthesis. * P < 0.05 when compared to control.
3.C
.
•
:Control
/
•
:IOOuM
/
*
(6) Ach/IOuM
o :IOOuM Ach/IOuM
E S (7)
ES +
•
LL
I
B
El1
I
E2
I
E3
p
~
I
P2
Fig. 2. Effects of A N F on evoked [3H]NE release with Ach. Number of determinations are shown in parenthesis. • P
