B and C Types Natriuretic Peptides Modify Norepinephrine Uptake and Release in the Rat Adrenal Medulla

Peptides, Vol. 18, No. 10, pp. 1483–1489, 1997 Copyright © 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/97 $17.00 1 .00

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B and C Types Natriuretic Peptides Modify Norepinephrine Uptake and Release in the Rat Adrenal Medulla M. S. VATTA, M. F. PRESAS, L. G. BIANCIOTTI, M. RODRIGUEZ-FERMEPIN, R. AMBROS AND B. E. FERNANDEZ Ca´tedras de Fisiologı´a y Fisiopatologı´a (PROSIVAD-CONICET), Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires. Argentina Received 21 April 1997; Accepted 14 July 1997 VATTA, M. S., M. F. PRESAS, L. G. BIANCIOTTI, M. RODRIGUEZ–FERMEPIN, R. AMBROS AND B. E. FERNANDEZ. B and C types natriuretic peptides modify norepinephrine uptake and release in the rat adrenal medulla. Peptides 18(10) 1483–1489, 1997.—We have previously reported that atrial natriuretic factor (ANF) modulates adrenomedullar norepinephrine (NE) metabolism. On this basis, the aim of the present work was to study the effects of B and C types natriuretic peptides (BNP and CNP) on the uptake, intracellular distribution and release of 3H-NE. Experiments were carried out in rat adrenal medulla slices incubated ‘‘in vitro.’’ Results showed that 100 nM of both, CNP and BNP, enhanced total and neuronal NE uptake. Both peptides (100 nM) caused a rapid increase in NE uptake during the first minute, which was sustained for 60 min. NE intracellular distribution was only modified by CNP (100 nM), which increased the granular fraction and decreased the cytosolic pool. On the other hand, spontaneous as well as evoked (KCl) NE release, was decreased by BNP and CNP (50 and 100 nM for spontaneous release and 1, 10, 50 and 100 nM for evoked output). The present results suggest that BNP and CNP may regulate catecholamine secretion and modulate adrenomedullary biological actions mediated by catecholamines, such as blood arterial pressure, smooth muscle tone, and metabolic activities. © 1997 Elsevier Science Inc. B-type natriuretic peptide Adrenal medulla

C-type natriuretic peptide

Norepinephrine uptake

THE natriuretic peptide family is composed of three peptides: atrial natriuretic factor (ANF), brain natriuretic peptide or B type natriuretic peptide (BNP) and C type natriuretic peptide (CNP). ANF is a 28 amino-acids peptide synthetized and released by mammalian atrial cardiocytes in response to atrial stretch (5,19). It induces diuresis and natriuresis, smooth muscle relaxation (3,22,29) and inhibits the release of several hormones such as aldosterone, vasopressin, prolactin, and CRF (1,3,7,27,29) and neurotransmitters (1,21,29,37). BNP which was originally evidenciated in porcine brain, is in fact mainly distributed in the cardiac atria of the human, rat and dog (11,26,28). Its aminoacid sequence is less conserved across the different species than that of ANF (13,26). BNP shares most of ANF biological actions such as cardiocirculatory regulation and diuresis and natriuresis (15,16,26). On the other hand, CNP is a 22 amino-acids peptide widely distributed in the central nervous system of different species (14,32). CNP also induces smooth muscle relaxation, however it does not cause diuresis and natriuresis as the other natriuretic peptides (4,30). We have previously reported that ANF modulated noradrener-

Norepinephrine release

gic neurotransmission, in the rat adrenal medulla, since it increased NE uptake and reduced NE release (23,39). Provided that CNP and BNP seem to be involved in most of the biological actions which are also modulated by ANF, we considered it of interest to study whether CNP and BNP regulated norepinephrine metabolism in the adrenal medulla of the rat. METHOD

Animals Male Wistar rats (250 –300 g) from the Patho-physiology Department, Faculty of Pharmacy & Biochemistry, UBA, Argentina, were housed under controlled conditions (22-24°C; 12-h light cycle, starting at 7:00 h). Animals were maintained ad libitum on Purina Chow and tap water. Drugs and Solutions The drugs used in this study were NE-HCl DL[7-3H(N)] (New England Nuclear, Boston, MA, USA) of 15 Ci/mmol of

Requests for reprints should be addressed to: Catedra de Fisiopatologia, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Junin 956-5to Piso, 1113 Buenos Aires, Argentina. E-mail: [email protected]

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FIG. 1. Effects of CNP (A) and BNP (B) on total 3H-NE utake. The columns are the means, and the vertical bars are the SEMs. The ANOVA and the ‘‘t’’ test modified by Bonferroni were used for statistical analysis. The asterisks indicate the statistical difference between control and treated groups (*, p , 0.05). Number of experiments, figure A (n 5 6-12) and figure B (n 5 7-10).

specific activity (1 Ci 5 37 GBq); rat BNP-32, Hydrocortisone (HC), Pargyline and Tropolone (Sigma Chemical Co., St. Louis, MO, USA); CNP (1-22) (Peptide Institute Inc., Osaka, Japan) and Cocaine Chlorohydrate (CO) (Supplied by the Toxicology Department, Faculty of Pharmacy & Biochemistry, UBA, Argentina); Liquid scintillation cocktail (Ultima Gold, Packard Inst. Co., CT, USA). Incubation medium: Standard Krebs bicarbonate solution (KBS) of the following composition (mM): NaCl 118; KCl 4.7; MgCl2 1.2; NaH2PO4 1.0; CaCl2 2.5; EDTA-Na 0.004; Dextrose 11.1; NaHCO3 25.0; Ascorbic acid 0.11. Experimental Procedures All experiments were carried out in vitro. Total 3H-NE uptake. Animals were sacrified by decapitation between 10:00 and 12:00 AM to avoid circadian changes. The adrenal glands were quickly removed and medullae were immediately dissected by decortication, cooled and weighed. Slices of about 1 mm were cut and then transferred into a glass tube (15.8 6 0.3 mg adrenal medulla slices/tube) with a mesh of nylon fitted at the bottom to allow free interchange with the medium. The tissues were placed in a Dubnoff incubator and

VATTA ET AL. pre-incubated for 15 min at 37°C, pH 7.4 and bubbled with carbogen (95% O2 1 5% CO2) under continued shaking with 2 ml of KBS. To avoid NE catabolism, monoamine oxidase (MAO) and cathecol O-methyl transferase (COMT) activities were inhibited for 30 min before and during the incubation period with 100 mM pargyline and 100 mM tropolone, respectively. In the incubation period, adrenal medulla slices were labelled with 1,25 mCi/ml of 3H-NE for 30 min in the absence (control group) or in the presence of BNP or CNP (1, 10 and 100 nM) (experimental groups). The tissues were washed for 5 min with 2 ml of KBS and then homogenized with 2.5 ml of 10% trichloroacetic acid and centrifuged at 27.000 g and 4°C for 15 min (Sorvall Superspeed RC2-B). Tritium activity was measured in supernatants by usual scintillation counting methods (Packard-PRIAS 240CL/D). Time course of 3H-NE uptake. Threshold concentrations of BNP and CNP were chosen according to total NE uptake study. The same protocol as described above for total 3H-NE uptake was performed. NE uptake was determined at 1, 5, 10, 30 and 60 min of incubation in the control and in BNP or CNP groups(both 100 nM). Neuronal and non-neuronal uptake. Diverse authors have reported that neuronal uptake did not differ in reserpined tissues when compared with non-reserpined tissues (8,34). Therefore, experiments were carried-out in non-reserpined tissues. Adrenal medulla slices were submitted to the same experimental procedures described for total NE uptake determination except that neuronal (uptake 1) and non-neuronal (uptake 2) were inhibited for 30 min before and during the incubation period with 10 mM CO (inhibits neuronal NE uptake) or 100 mM HC (inhibits extraneuronal NE uptake) (10,35). The following groups were studied: a) control, and incubated with, b) HC; c) HC 1 100 nM BNP; d) HC 1 100 nM CNP; e) CO; f) CO 1 100 nM BNP and g) CO 1 100 nM CNP. Intracellular distribution of 3H-NE. The tissues were submitted to the same experimental procedures as described for total NE uptake determination except that the tissues were homogenized with 0.32 M sucrose and then ultracentrifuged (Sorvall Ultracentrifuge OTD-55B) at 100.000 g for 90 min at 4°C to separate the cytosolic and granular fractions (23). Both fractions were deproteinized with 10% trichloroacetic acid and centrifuged for 15 min at 27.000 g. Trytium activity was determined in the supernatants. Neuronal 3H-NE release. The same protocol as described for total NE uptake determination was followed, but 30 min before and during the incubation period the uptake 2 (nonneuronal uptake) was inhibited with 100 mM HC. After the incubation period, the adrenal medullae were washed for 30 min with 25 ml of KBS (in six consecutive washing periods of 5 min), to avoid extracellular and extraneuronal release. The tissues were incubated for 10 min and two consecutive samples of the medium were collected every 5 min The first sample corresponded to the basal period and the second sample belonged to the experimental period. Five min before and during the basal and experimental periods the neuronal uptake was inhibited with 30 mM CO. Adrenal medullae were randomly divided into two groups for: a) the determination of spontaneous neuronal 3H-NE release in control group and groups treated with BNP or CNP (1, 10 and 100 nM) and b) the determination of evoked neuronal release of 3H-NE in control group and groups treated with 25 mM KCl or 25 mM KCl plus BNP or CNP (10, 100 pM and 1, 10 and 100 nM). The volumes (2 ml) of all the samples were reduced to 0.2 ml in a vacuum stove

BNP, CNP, AND NE UPTAKE AND RELEASE

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FIG. 2. Effects of CNP (A) and BNP (B) on the time course of 3H-NE uptake. Filled squares: control group; Filled triangles: 100 nM CNP; Filled circles: 100 nM BNP. The vertical bars are the SEMs. The ANOVA and the ‘‘t’’ test modified by Bonferroni were used for statistical analysis. The asterisks indicate the statistical difference between the control and treated groups (*, p , 0.05). Number of experiments, figure A (control 5 7; CNP 5 5) and figure B (control 5 8; BNP 5 6).

(National Appliance Co., Model 5831) and 3H activity was determined by usual scintillation counting methods. Analysis of results NE uptake is expressed as dpm/g of the fresh tissues 6 S.E.M.. Intracellular distribution of NE is expressed as the percentage 6 S.E.M. of total uptake corresponding to cytoplasmatic or granular fractions. The results of neuronal NE release are expressed as the factor above basal release of 3H 6 S.E.M. One way analysis of variance (ANOVA) and the ‘‘t’’ test modified by Bonferroni were used for statistical analysis (41). P values of 0.05 or less were considered statistically significant.

RESULTS

Total 3H-NE uptake Concentration response studies were performed to determine the threshold concentrations of CNP and BNP which modified total NE uptake. Fig. 1A and 1B illustrate that the threshold concentration for both peptides was of 100nM. Concentrations of 1, 10 and 50 nM had no effect on the amine uptake. Time course of 3H-NE uptake Results showed that BNP as well as CNP (100 nM) increased NE uptake in the first min of the incubation period which was

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VATTA ET AL. study the effects of both BNP and CNP on neuronal NE output. Results showed that 50 and 100 nM of both, BNP and CNP reduced NE neuronal release, whereas 1 and 10 nM did not affect the amine secretion (Fig. 5A and 5B). In addition, NE output evoked by 100 mM KCl, was decreased by 10, 50 and 100 nM BNP and CNP (Fig. 6A and 6B). Both CNP and BNP, modified spontaneous as well as evoked NE release in a concentration dependent way. DISCUSSION

Present results provide evidence for the role of BNP and CNP in the modulation of the biological actions mediated by adrenomedullary catecholamines. Both, ANF and BNP immunoreactivity have been demonstrated in the adrenal medulla (17). Furthermore, Komatsu et al. (14), reported that CNP immunoreactivity was not only distributed in various regions of the CNS, but also in several other tissues including the adrenal medulla. In addition, the presence of CNP mRNA has been described in chromaffin tissue (20). Two types of natriuretic factor receptors have been identified in target tissues: two different guanylate cyclase linked receptor (NPR-A and NPR-B) that appear to mediate most of the natriuretic peptides biological effects and a non-coupled guanylate cyclase or clearance receptor (NPR-C) (3,26). The latter seems to be involved in the

FIG. 3. Effects of CNP (A) and BNP (B) on neuronal and extraneuronal 3H-NE uptake. The columns are the means, and the vertical bars are the SEMs. The ANOVA and the ‘‘t’’ test modified by Bonferroni were used for statistical analysis. One asterisk indicates the statistical difference respect to the control group (*, p , 0.05) and two asterisks indicate the statistical difference respect to the 100 uM HC group. Number of experiments: figure A (n 5 6-7) and figure B (n 5 6-8).

sustained up to 60 min (Fig. 2A and 2B). Both peptides showed quite similar 3H-NE uptake time course curves. Neuronal and extraneuronal 3H-NE uptake The aim of this experimental approach was to determine whether the total NE uptake produced by BNP and CNP was due to an increase of either neuronal and/or extraneuronal NE uptake. The effects of threshold concentrations of BNP and CNP were studied in the presence of 10 mM CO or 100 mM HC. Results showed that both peptides increased neuronal uptake but did not affect the remanent extraneuronal uptake of the amine (Fig. 3A and 3B). Intracellular distribution of 3H-NE Figure 4A illustrates that 100 nM BNP did not modify the intracellular distribution of NE. Conversely, 100 nM CNP diminished the cytosolic pool, increasing the granular fraction of NE (Fig. 4B). Neuronal release of 3H-NE Concentration response studies were performed in rat adrenal medulla slices after labelling NE stores with 3H-NE for 30 min to

FIG. 4. Effects of CNP (A) and BNP (B) on intracellular distribution of NE. Open column: control; down-hatched column: 100 nM CNP; uphatched: 100 nM BNP. The columns are the means, and the vertical bars are the SEMs. The ANOVA and the ‘‘t’’ test modified by Bonferroni were used for statistical analysis. The asterisks indicate the statistical difference between the control and treated groups (*, p , 0.005). Number of experiments, figure A (n 5 7) and figure B (n 5 6).

BNP, CNP, AND NE UPTAKE AND RELEASE

1487 granular and the cytosolic pools of NE. However, CNP enhanced NE content into the granular pool, decreasing the cytosolic fraction. The effect of CNP may result from the higher increase of NE uptake, which in turn may displace the cytosolic content of NE towards the granular pool. This process involves an increase in the activity of the vesicular amine transporter, dependent on Mg21 ATPase activity (31). The difference of CNP and BNP response could be due to the fact that CNP enhancement of neuronal NE uptake was greater than that induced by BNP (56% vs 20%). Another possibility is that CNP increases the activity of the vesicular amine transporter, as suggested for ANF (39). Both BNP and CNP slighlty reduced spontaneous NE release. The same behaviour was observed in the central nervous system (40). However, when NE output was evoked by 25 mM KCl the reduction was more important, which supports the role of the natriuretic peptides as modulators of biological responses specially when stimulated. For example, the reduction of blood arterial pressure induced by the natiuretic peptides is slight, however in the presence of angiotensin II the reduction is considerebly relevant (3). Furthermore the reduction of sodium reabsorption in the kidney induced by ANF is evident in the presence of angiotensin II (3). In other words the effects of the natriuretic peptides seem to play a physiological role when the system is stimulated. A great

FIG. 5. Effects of CNP (A) and BNP(B) on the spontaneous neuronal release of 3H-NE. The columns are the means, and the vertical bars are the SEMs. The ANOVA and the ‘‘t’’ test modified by Bonferroni were used for statistical analysis. The asterisks indicate the statistical difference between the control and treated groups (*, p , 0.005; **, p , 0.025; ***, p , 0.05). Number of experiments, figure A (n 5 6-10) and figure B (n 5 6-8).

regulation of circulating natriuretic peptide levels, although recent reports suggest that this type of receptor would mediate biological responses through the activation of phosphoinositide hydrolysis and/or the inhibition of adenylate cyclase (1,9,18). A new natriuretic peptide receptor named natriuretic peptide receptor-D (NPR-D) has been cloned and characterized in the eel brain (12). Both NPR-A and NPR-B have been identified in the adrenal medulla, however NPR-D has not been found in this tissue (12). All natriuretic peptides are able to bind to both types of receptors, although with different affinity. ANF is a more selective ligand for NPR-A receptor than BNP or CNP. Conversely, CNP displays the highest selectivity binding for NPR-B receptor (CNP.ANF.BNP) (13,33). Present results showed that, although 100 nM was the threshold concentration for both BNP and CNP to increase NE neuronal uptake, the effect of CNP was higher than that of BNP (56% vs 20%). Nevertheless, previous works (39) showed that 10 and 100 nM ANF induced a sharper increase (60% and 123% respectively). These results suggest that both, BNP and CNP, increase neuronal NE uptake, likely through an increase in the activity and/or the synthesis of NE reuptake transporters. Furthermore, 100 nM ANF produces a higher increase of NE uptake than equimolar concentrations of CNP and BNP (2.2 and 6.2 times, respectively). Although, BNP did not affect the equillibrium between the

FIG. 6. Effects of CNP (A) and BNP (B) on the neuronal 3H-NE release evoked by 100 mM KCl. The columns are the means, and the vertical bars are the SEMs. The ANOVA and the ‘‘t’’ test modified by Bonferroni were used for statistical analysis. The asterisks indicate the statistical difference between the 100 KCl group and treated groups (*, p , 0.001; **, p , 0.005; ***, p , 0.01). Number of experiments, figure A (n 5 6-10) and figure B (n 5 5-8).

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body of evidence show that the natriuretic peptides would counterbalance the action of vasopressor systems, specially in pathophysiological situations. Fifty and 100 nM BNP induced a decrease of 12,8% and 16,3% respectively while 50 and 100 nM CNP caused a reduction of 18,6% and 24% respectively in NE spontaneous output. On the other hand, 1, 10, 50 and 100 nM BNP induced a decrease of 33,3%, 39%, 46,3% and 51,8% respectively, in evoked NE secretion, while 1, 10, 50 and 100 nM CNP caused a reduction of 27,3%, 34,8%, 36,4% and 43,9% respectively, in evoked NE output. These results show that both peptides affected NE output in a concentration dependent fashion. In a previous work, we demonstred that 10 nM ANF reduced both, spontaneous and evoked NE release (27% and 58%, respectively) (39). NE secretion is a calcium dependent complex mechanism that involves several cytoeskeleton proteins such as actin, fodrin, a-actin and scinderin (36). The actin filament network restrains chromaffin granules from interacting with exocytotic sites on the plasma membrane. When cells are stimulated, Ca21 influx increases and detaches chromaffin granules, bounds to actin filaments and activates proteins that serve actin filaments. This form is of low viscosity and permits the chromaffin granules exocytosis (36). BNP and CNP may affect any step of NE secretion, such as Ca21 influx, thus affecting NE secretion. A futher possibility is that these peptides may inhibit the nicotinic receptor, as it has been reported for ANF (2) or stimulate outward potassium channels. According to the present results BNP and CNP showed quite similar behaviour as compared to ANF, except that BNP did not alter intracellular distribution of the amine (39). The quantitative differences observed among the effects of the three peptides of the natriuretic family may be probably related to the different affinity of the peptides towards NPR-A and NPR-B receptors (13,33) in the adrenal medulla. Previous reports showed that, in the central nervous system,

natriuretic peptides (ANF, BNP, and CNP) enhance neuronal NE uptake and reduce spontaneous as well as evoked neuronal release of the amine diminishing the amount of NE in the synaptic space (6,37,40), thus reducing noradrenergic activity at the presynaptic nerve ending level. Moreover, similar effects were reported for ANF in rat adrenal medulla (39). Present results show that BNP as well as CNP exert effects on norepinephrine uptake and release in the adrenal medulla which are similar to those observed for ANF. On the other hand, natriuretic peptides have the opposite effects of angiotensin II and III. These include aldosterone and vasopressin secretion, proximal tubular sodium and water reabsorption, smooth muscle activity and NE uptake and release (3,7,24,25,37–39). The natriuretic peptides-angiotensins-NE interactions may be involved in diverse physiological and several patho-physiological status modulated at the adrenal medulla level. The presence of CNP immunoreactivity in plasma has not been reported (4). Furthermore the content of CNP in the adrenal medulla is very low. However the existance of CNP RNAm in the adrenal medulla suggests local synthesis of the peptide (20). These findings suggest that CNP effects in the adrenal medulla are likely to be paracrine dependent on local synthesis of the peptide. In addition the events which stimulates CNP synthesis in the adrenal medulla remain unknown. In conclusion, the present results suggest that the three members of the natriuretic family, BNP, CNP and ANF may regulate several biological actions regulated by adrenomedullary catecholamines, such as blood arterial pressure, smooth muscle tone and diverse metabolic activity. ACKNOWLEDGEMENTS

This work was supported by grants of the Universidad de Buenos Aires (UBACyT:FA 134).

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