Effects of atrial natriuretic factor on norepinephrine release in the rat hypothalamus

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

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A N F (7)

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(7)

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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

.

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/

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:IOOuM

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(6) Ach/IOuM

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E S (7)

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Fig. 2. Effects of A N F on evoked [3H]NE release with Ach. Number of determinations are shown in parenthesis. • P<0.05 when compared to control; **P<0.05 when compared to 100 #M Ach/10 #M ks.

175

Statistical analysis Results are expressed as the fractional release of [3H]NE S.E.M. in each sample considering the activity of the basal samples as 1, since the ratio between the basal samples of each of the different experiments is 1. Fracional release of every sample was calculated as the ratio between [3H]NE release in each experimental or postexperimental period and basal output of [3H]NE. Two factor design with interaction-ANOVA and the t-test modified by Bonferroni were used for statistical analysis [27]. P<0.05 or less were considered statistically significant.

Results

Effects of ANF on spontaneous and evoked [SHINE release 10 nM ANF decreased spontaneous NE output in the first experimental period (3 rain), while no modifications were observed in the second and third experimental periods (6 and 9 min). Conversely, 50 nM ANF also decreased NE output in the first as well as the second experimental periods (3 and 6 min) but no modifications were observed in the third experimental period (9 min). In both cases, 10 and 50 nM ANF, the release of NE reached basal levels in the two post-experimental periods (12 and 15 min). On the other hand, 100 nM ANF reduced NE release in all experimental periods (3, 6 and 9 min) and the first post-experimental period (12 min). NE output reached basal levels in the second post-experimental period (15 min) (Fig. 1). In order to observe the effects of 10 nM ANF on evoked NE release, experiments were performed in the presence of Ach/Es. Evoked NE output was reduced by 10 nM ANF in the three experimental periods and the two post-experimental periods (Fig. 2).

•

:Control

o : CFM

1.O lxl Z i

T

•

(8)

(7)

:CFM+10nM

ANF(7)

0.9

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t_

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0.6 0.5

0.4 L

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i

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Fig. 3. Effects of A N F on [3HINE release in CFM. Number of determinations are given in parenthesis. * P < 0.05 when compared with control.

176

~ 2.e

*

i / I k

1/ /

• :IO0uM Ach/IOuM Es (6) o :IO0uM Ach/lOuM Es+CFM (6) II:lO0uM Ach/IOuM Es+CFM÷

~ lo..AnF(6) \

2.C

-~

1.6

s

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0.8

0.4 I

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Fig. 4. Effects of A N F on [3H]NE release in CFM/Ach. Number of determinations are shown in parenthesis. *P<0.05 when compared to 100 #M Ach/10/~M Es.

Effects of ANF on NE release in CFM

Fig. 3 illustrates that basal [3H]NE output decreased in a CFM in all experimental periods (3, 6 and 9 min), while no modifications, compared to basal values, were observed in the first and second post-experimental periods. When 10 nM ANF was added to the CFM, the amine release was not altered in the studied periods compared with the CFM. ANF (10 nM) was unable to modify the secretory response promoted by a CFM in the presence of 100 #M Ach/Es, in both experimental and post-experimental periods (Fig. 4).

•

ILl

1.0

~ |

'

:Control

(7)

o : IOOuM

DTZ (7)

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Fig. 5. Effects of A N E on [3H]NE release in the presence of DTZ. Number of cases are given in parenthesis. * P < 0.05 when compared with control.

177

3.0

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.L

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Fig. 6. Effects of ANF on evoked [3H]NE release with Ach in the presence of DTZ. Number of determinations are shown in parenthesis. *P<0.05 when compared to 100 #M Ach/10 /~M Es; **P<0,05 when compared to 100/IM Ach/10 #M Es + 100 #M DTZ.

Effects of A N F on NE output in the presence of a calcium channel blocker (DTZ) 100/~M DTZ decreased NE release in the first experimental period (3 min) while no modifications were observed at second and third experimental periods and two post-experimental periods (6, 9, 12 and 15 min). The addition of 100 #M ANF to the standard Krebs solution containing DTZ induced no modifications neither in the experimental periods nor in the post-experimental periods (Fig. 5). Fig. 6 shows that 10/~M DTZ decreased NE secretion evoked by 100/~M Ach/10 /~M Es in all experimental periods (3, 6 and 9 min), and post-experimental periods (12 and 15 min). When 10 nM ANF was added to Krebs solution with Ach/Es plus DTZ, the atrial factor decreased NE release in the third experimental period (9 min) and the first post-experimental period (12 rain).

Discussion

Central catecholamines of encephalic regions such as hypothalamus, medulla oblongata and other brain areas, are involved in blood arterial pressure regulation and neuroendocrine processes [22-24]. Vasoactive peptides such as ANG II and ANF may enter into the central nervous system through the blood brain barrier close to the circumventricular organs (subfornical organs, organum vasculosum lamina terminalis, area postrema) [19-21]. ANG II exerts central direct hypertensive effects since it stimulates the anteroventral region of the third ventricle (AV3V Brody area) [28]. In addition, it has also indirect

178 effects since it increases vasopressin output and controls central sympathetic activity by regulating catecholamine metabolism [20,29]. The existence of A N F binding sites have been described in several areas of the central nervous system closely related to cardiocirculatory centers [30]. Central A N F receptors may be involved in the regulation of blood arterial pressure [21,31], cerebroespinal fluid production [32] and the hypothalamic secretion of pituitary inhibitory or releasing factors [30]. Inmunoreactive A N F high density areas of the central nervous system correlate well with high density areas of catecholamines and A N G lI [29,30,33]. Moreover, A N F receptors are located in the same areas of the central nervous system, being these areas closely related to the regulation of blood arterial pressure [33 ]. A natriuretic factor with similar effects to those of circulating ANF, with homologous aminoacidic sequence but different molecular weight has been characterized in the central nervous system. The localization in discrete central areas of the brain, suggests its local synthesis which has been supported by the finding of A N F - m R N A in extracts of cerebellum, hypothalamus, thalamus and other brain regions [34], and by the presence of different types of natriuretic factors (A, B and C) [35]. We have previously reported that A N F increases neuronal N E uptake and modifies N E intracellular distribution in the central nervous system [16,17]. We have also observed the existence of A N F - A N G II and A N F - A N G III interactions on N E uptake in the same central structures [15,17,18]. On the other hand, Chartier and Schiffrin [36] have demonstrated that in isolated rat adrenal glomerulosa cells, A N F blocked aldosterone secretion and synthesis, previously stimulated by A N G II, A C T H and potassium despolarizing solutions. They suggested that A N F may act at least in part by interfering with calcium entry. Present results show that A N F reduces spontaneous neuronal N E release in the rat hypothalamus, being this effect concentration related. When N E output was induced by Ach solution, similar results were observed, 10 nM A N F reduced the release of the amine. The reduction or N E output was different depending upon the concentration of A N F used: 10 nM ANF: 21~,; (El); 50 nM ANF: 22 and 15% (El and E2, respectively) and 100 nM ANF: 31, 25, 25 and 25~o (El, E2, E3 and P1, respectively). It is important to point out that the reduction of N E output produced by A N F (10 nM) was higher when the release was induced by Ach (70, 63 and 59% in El, E2 and E3, respectively). The study of A N F effects on calcium dependent NE releasing mechanisms showed the amine output diminished when the incubation medium was deprived of calcium (between 49 and 42}0 with respect to the spontaneous secretion). When A N F was added to this medium, the release of N E fell to similar levels of those of the C F M (CFM: between 49 and 42}0 vs. C F M + ANF: between 46 and 43}0). As A N F alters evoked N E release more than the basal N E secretions, the effects of this peptide in a C F M were investigated in the presence of Ach. Results showed that no modifications were induced by the addition of A N F in a CFM-Ach medium. When the tissues were incubated in the presence of a calcium channel blocker as DTZ, A N F was unable to modify the secretory response produced by D T Z on spontaneous N E release. The addition of A N F to a medium with D T Z plus Ach enhanced

179

the inhibition of DTZ on evoked N E output (DTZ + Ach: 59~o vs. DTZ + Ach + ANF: 71 ~o). This additive effect of ANF and DTZ on Ach evoked NE release was prolonged up to the first post-experimental period. Present results show that ANF diminished spontaneous as well as Ach induced NE release although the ANF impact was greater on the evoked release of the amine. The effects of ANF on N E output was not evident when the incubation medium was deprived of calcium. A N F and DTZ evoked an additive effect when N E release was induced by Ach. Our results support the hypothesis that ANF mechanisms on NE output is related to calcium, being its action probably exerted on the calcium exocytotic process of N E release. In addition, ANF and DTZ evoked an additive effect, indicating a role of A N F over calcium metabolism thus indirectly affecting central catecholamine output. AV3V vasodepressor and ~2 vasopressor areas located in the hypothalamus are intimately related to the regulation of blood arterial pressure. Central sympathetic activity modulation produced by ANF and A N G II and the interaction between these peptides occurring at this level suggest that both peptides may play an important role in the regulation of blood arterial pressure and the desequillibrium between the A N F A N G II interaction and sympathetic activity would affect normal cardiovascular functionality.

Acknowledgements This work was supported by grants from the Consejo Nacional de Investigaciones Cientificas y Tecnicas of Argentina (CONICET). We thank Roemmers Lab. Argentina, for the generous supply of Diltiazem.

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