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B and C types natriuretic peptides modulate norepinephrine uptake and release in the rat hypothalamus
REGULATORY
PEPTIDES
ELSEVIER
Regulatory Peptides 65 (1996) 175-184
B and C types natriuretic peptides modulate norepinephrine uptake and release in the rat hypothalamus M.S. Vatta *, M. Presas, L.G. Bianciotti, V. Zarrabeitia, B.E. Fernfindez Cdtedras de Fisiopatologia and Fisiologfa, Programa de Sistemas Vasodepresores - Consejo Naeional de lnvestigaciones Cientfficas y Tdcnicas (PROS1VAD-CONICET), Facultad de Farmacia y Bioqufmica, Universidad de Buenos Aires, Buenos Aires, Argentina Received 8 February 1996; revised 11 May 1996; accepted 20 May 1996
Abstract
We previously reported that atrial natriuretic factor (ANF) regulates catecholamine metabolism in the central nervous system. ANF, B and C types natriuretic peptides (BNP and CNP) also play a regulatory role in body fluid homeostasis, cardiovascular activity and hormonal and neuro-hormonal secretions. The aim of the present work was to investigate BNP and CNP effects on the uptake and release of norepinephrine (NE) in rat hypothalamic slices incubated in vitro. Results showed that BNP (100 riM) and CNP (1, 10 and 100 nM) enhanced total and neuronal [3H]NE uptake but did not modify non-neuronal uptake. BNP (100 nM) and CNP (1 nM) caused a rapid increase in NE uptake (1 rain), which was sustained for 60 min. BNP (100 nM) did not modify the intracellular distribution of NE; however, 1 nM CNP increased the granular store and decreased the cytosolic pool of NE. BNP (100 nM) and CNP (1, 10 and 100 riM), diminished spontaneous NE release. In addition, BNP (1, 10, 100 nM) and CNP (1, 10 and 100 pM, as well as 1, 10 and 100 nM) reduced NE output induced by 25 mM KC1. These results suggest that BNP and CNP may be involved in the regulation of several central as well as peripheral physiological functions through the modulation of noradrenergic neurotransmission at the presynaptic neuronal level. Present results provide evidence to consider CNP as the brain natriuretic peptide since physiological concentrations of this peptide (pM) diminished NE evoked release. Keywords: Neuronal norepinephrine uptake; Non-neuronal norepinephrine uptake; Norepinephrine intracellular distribution; Central nervous system; Atrial natriuretic factor
1. Introduction
Atrial natriuretic factor or A type natriuretic factor (ANF) originally discovered by de Bold et al. [1], is synthesized, stored and released by mammalian atrial cardiocytes in response to atrial stretch [2,3]. Several reports have demonstrated that, in fact, a family of natriuretic peptides exists: ANF, B type natriuretic peptide (BNP) and C type natriuretic peptide (CNP). Although BNP was initially identified in porcine brain, this peptide is more abundant in cardiac atriae and ventricles than in the central nervous system (CNS) [4-6]. Its amino acid sequence and biological actions are very similar to those of ANF. In the human and pig, a 32-amino-acid peptide is the circulating form of BNP [7], while in rodents
* Corresponding author. C~tedra de Fisiopatologla, Facultad de Farmacia y Bioqulmica, Universidad de Buenos Aires. Jun~n 956 - 5to piso, (1113) Buenos Aires, Argentina.
the 45-amino-acid sequence is the mature form of BNP [8]. Circulating BNP may play a role in regulating water and electrolyte balance and cardiovascular homeostasis [6,7,9,10]. The newest member of the natriuretic family is CNP, which is a 22-amino-acid peptide that has been identified in the CNS, kidney and intestine of pig, human and rat [11,12]. Burnett and co-workers have demonstrated that the intravenous administration of CNP causes a marked decrease in blood arterial pressure, cardiac filling pressure and cardiac output without natriuresis [13,14]. CNP circulates in low picomolar concentrations and is a potently vasoactive in vivo, suggesting a potential role in the regulation of vascular tone [14,15]. CNP immunoreactivity is present in human endothelial cells and it could be an important component of an autocrine endothelium-derived vasomodulatory system [ 13,16,17]. Two types of natriuretic factor receptor have been identified in target tissues: two different guanylate cyclase linked receptors that appear to mediate most of the biologi-
0167-0115/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PI1 S01 6 7 - 0 1 1 5 ( 9 6 ) 0 0 0 9 0 - 0
M.S. Vatta et al./Regulatory Peptides 65 (1996) 175-184
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cal effects (ANFR-A and ANFR-B) and the non-guanylate coupled receptor or clearance receptor (ANFR-C) which appears to regulate the natriuretic factors circulating levels [2,7]. Nevertheless it has been demonstrated that in several tissues the latter may be biologically active. It does not signal through activation of guanylyl cyclase, but may function through intermediate G-protein to inhibit adenylate cyclase or activate phosphoinositide pathway [7,18,19]. Both ANFR-A and ANFR-B are widely distributed in the CNS and other peripheral tissues [7,18,19]. We have previously reported that, in the CNS of the rat, ANF increases neuronal norepinephrine (NE) uptake and NE content into the granular fraction [20-22]. In addition, it diminishes spontaneous as well as evoked NE release [23,24] and NE turnover (unpublished data). In view of ANF effects on noradrenergic neurotransmission, the presence of BNP and CNP immunoreactivity in 45-
several encephalic regions such as hypothalamus and ANF-A and ANF-B receptors distribution in the CNS, we considered it of interest to study BNP and CNP effects on: (a) total [3H]NE uptake; (b) time course of [3H]NE uptake; (c) neuronal and non-neuronal [3H]NE uptake; (d) intracellular distribution of [3H]NE; and (e) spontaneous and evoked neuronal release of [3 H]NE.
2. Materials and methods 2.1. Animals
Wistar strain male rats weighing between 250-300 g (From the Pathophysiology Department, Faculty of Pharmacy & Biochemistry, UBA, Buenos Aires, Argentina) were used in all experiments. The rats were housed in steel
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M.S. Vatta et al./Regulatot T Peptides 65 (1996) 175-184
2.3. Experimental procedures
cages and maintained at a temperature between 22-24°C in a controlled room with a 12 h light/dark cycle (light from 7:00 to 19:00 h). Animals were allowed free access to tap water and food (commercial rodents Purina chow).
All experiments were carried out in vitro.
2.2. Drugs and solutions The following drugs were used in the experiments: NE-HC1 DL [7-3H(N)] (New England Nuclear, Boston, MA, USA; of 15 C i / m m o l of specific activity; rat BNF-32 (Sigma, St. Louis, MO, USA); CNF (1-22) (Peptide Institute, Osaka, Japan); hydrocortisone (HC) and pargyline (PRG) (Sigma) and cocaine chloridrate (Co) (supplied by the Toxicology Department, Faculty of Pharmacy & Biochemistry, UBA). Incubation medium: Standard Krebs bicarbonate solution of the following composition (in mM): NaC1 118; KC1 4.7; MgC12 1.2; NaH2PO 4 1.0; CaC12 2.5; EDTA-Na 0.004; dextrose 11.1; NaHCO 3 25.0; ascorbic acid 0.11.
2.3.1. Total [ SH]NE uptake Animals were decapitated between 10:00 and 12:00 a.m. to avoid circadian changes. Brains were quickly removed and hypothalami were immediately dissected, cooled and weighed. Slices were cut and then transferred into a glass tube with a mesh of nylon fitted at the bottom to allow free interchange with the medium. Slices were placed in a Dubnoff incubator and pre-incubated for 15 min at 37°C (pH 7.4) and bubbled with carbogen (95% O2/5% CO 2) under continued shaking with 2 ml of Krebs bicarbonate solution. Monoamine oxidase (MAO) activity was inhibited by the addition of PRG (100 mM) for 30 rain. NE stores were labelled during the 30 min incubation period [25] with 1.25 mCi/ml of [3H]]NE in the absence (control group) or in the presence of BNP (1, 10 and 100
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M.S. Vatta et al. / Regulatory Peptides 65 (1996) 175-184
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nM) or CNP (10 and 100 pM; 1, 10 and 100 nM) (experimental groups). During this period MAO activity was also inhibited with 100 mM PRG. Slices were washed for 5 min with 2 ml of Krebs bicarbonate solution and were then homogenized with 2.5 ml of 10% trichloroacetic acid and centrifuged at 27000 × g and 4°C for 15 min (Sorvall Superspeed RC2-B). Tritium activity was determined in supernatants by usual scintillation counting methods (Packard-PRIAS 240CL/D).
scribed for total [3H]NE uptake determination. NE uptake was determined at 1, 5, 10, 30 and 60 min. The following groups were studied: (a) control slices; (b) slices incubated with 100 nM BNP; and (c) slices incubated with 1 nM CNP.
2.3.3. Neuronal and non-neuronal [3H]NE uptake Several authors have reported that neuronal NE uptake did not differ in reserpined tissues when compared with non-reserpined tissues [26,27]. Therefore, experiments were carried out in non-reserpined tissues. Hypothalamic slices were submitted to the same procedures as described for total NE uptake determination, except that neuronal and non-neuronal [3H]NE uptake were inhibited by 10 mM Co
2.3.2. Time-course of [~H]NE uptake Threshold concentrations of BNP and CNP were chosen according to total [3H]NE uptake study. Tissues were submitted to the same experimental procedures as de35-
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M.S. Vatta et al. / Regulatory Peptides 65 (1996) 175-184
and 100 mM HC, respectively. These inhibitions were carried out 30 min before and during the incubation period. The following groups were studied: (a) control and incubated with: (b) HC; (c) HC + 100 nM BNP; (d) HC + 1 nM BNP; (e) Co; (f) Co + 100 nM BNP; and (g) Co + 1 nM CNP.
2.3.4. Intracellular distribution of [YH]NE Hypothalamic tissues were submitted to the same experimental procedures as described for total NE uptake determination except that after the washing period, 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 (supernatant) and the granular (pellet) fractions [28]. Both pools were deproteinized with 10% trichloroacetic acid and centrifuged at 27000 × g for 15 min. Tritium activity was QO
179
measured in the supernatants of the following groups: (a) control and incubated with: (b) 100 nM BNP and (c) 1 nM CNP.
2.3.5. Neuronal [3H]NE release Hypothalamic slices were pre-incubated for 15 min at 37°C (pH 7.4) and bubbled with carbogen (95% 0 2 / 5 % CO2) under continuous shaking with 2 ml of Krebs bicarbonate solution. Then, MAO activity and extraneuronal uptake of NE were inhibited by the addition of 100 mM PRG and 100 mM HC, respectively, 30 min before and during the incubating period (30 min). In this last period, tissues were also labelled with [3H]NE. To avoid extracellular release and to obtain the equillibrium of the basal neuronal release, hypothalami were washed for 30 min with 25 ml of modified Krebs solution (in six consecutive washing periods of 5 min). The tissues were then incu-
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Fig. 4. Effects of BNP (a) a n d C N P (b) on [ 3 H ] N E intracellular distribution. Open column: control; down-hatched column: 100 nM BNP; up-hatched column: 1 nM CNP. Data are shown as mean + S E M (n = 6-7). * P < 0.05 compared with control.
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bated for 10 min and two consecutive samples of the medium were collected every 5 min. The first sample corresponded to the basal release period and the second sample belonged to the experimental period. Hypothalami were divided into groups for: (a) the determination of spontaneous neuronal [3H]NE release in control group and groups treated with BNP (1, 10, 100 nM) and CNP (10, 100 pM and 1, 10, 100 nM), and (b) the determination of evoked neuronal release of [3H]NE in control group and groups treated with 25 mM KC1 or 25 mM KC1 plus BNP (10, 100 pM and 1, 10, 100 nM) or CNP (10 and 100 fM; 1, 10 and 100 pM; 1, 10 and 100 nM). The volumes of all the samples were reduced to 0.2 ml in a vacuum stove (National Appliance, Model 5831) and 3H activity was measured by usual scintillation counting methods. 2.4. Analysis of results
NE uptake is expressed as d p m / g of the fresh tissues _+ SEM. Intracellular distribution of NE is expressed as the 1-
percentage + SEM 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 _ SEM. One-way analysis of variance (ANOVA) and the t-test modified by Bonferroni were used for statistical analysis [29]. P values of 0.05 or less were considered statistically significant.
3. Results 3.1. Total [3H]NE uptake
Concentration-response studies were carried-out using increasing concentrations of BNP and CNP in order to determine if these peptides modified total [3H]NE uptake. Fig. la and lb illustrate the threshold and subthreshold concentrations of BNP and CNP on NE uptake. Results showed that 1 and 10 nM BNP did not modify total [3H]NE uptake, whereas 100 nM BNP increased the amine
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M.S. Vatta et al. / Regulator)' Peptides 65 (1996) 175-184 uptake (Fig. la). On the other hand, l0 and 100 pM CNP failed to modify total NE uptake; however, 1, 10 and 100 nM C N P increased total [3H]NE uptake in a concentration-dependent fashion (Fig. lb).
181
3.3. Neuronal and non-neuronal [ 3H]NE uptake The aim of this study was determine if the increase observed in total NE uptake was due to an increase in either neuronal or non-neuronal [3H]NE uptake. When neuronal [3H]NE uptake was inhibited by 10 mM Co, the residual non-neuronal uptake was not modified by threshold concentrations of either BNP (100 nM) or CNP (1 riM). Nevertheless, when non-neuronal [3H]NE uptake was inhibited by 100 mM HC, the remaining neuronal NE uptake was increased by threshold concentrations of both BNP and CNP (Fig. 3a and b).
3.2. Time-course of [3H]NE uptake Fig. 2a and b show the modifications on NE uptake along the experimental period of 60 min. Threshold concentrations of both BNP (100 nM) and CNP (1 nM) increased total [3H]NE uptake in the first minute of the experimental period as compared to control time course. The curve of BNP on total NE uptake reached its highest level at 30 min and remained significantly higher than the control curve up to the end of the study (60 min) (Fig. 2a). On the other hand, CNP time-course showed a progressive increase in [3H]NE uptake (from 1 min to 60 min) reaching the maximum value at the end of the experimental period (Fig. 2b).
3.4. Intracellular distribution of [ 3H]NE Results showed that 100 nM BNP did not alter the intracellular distribution of NE (Fig. 4a). However, a threshold concentration of CNP (1 nM) diminished NE
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cytosolic pool content and increased NE vesicular pool content (Fig. 4B). 3.5. N e u r o n a l [ 3H ] N E release
Both BNP and CNP modified spontaneous, as well as evoked neuronal release of NE. 1 and 10 nM BNP, as well as 10 and 100 pM CNP did not modify spontaneous neuronal output of the amine. However, 100 nM BNP and 1, 10, 100 nM CNP diminished spontaneous neuronal NE secretion, in a concentration dependent fashion, as compared with the control group (Fig. 5a and b). On the other hand, Fig. 6a and b show that 1, 10, 100 nM BNP and 1, 10 and 100 pM as well as 1, 10 and 100 nM CNP decreased [3H]NE secretion evoked by a high potassium despolarizing solution (25 mM KC1). Conversely, 10 and 100 pM BNP as well as 10 and 100 fM CNP did not alter the evoked release of NE.
4. Discussion Hypothalamic and brain catecholamines are involved in the regulation of several biological actions such as cardiovascular activity, water and electrolyte balance, endocrine and neuroendocrine synthesis and secretion, thirst and apatite, etc. [30]. The existence of immunoreactive natriuretic factors and catecholamines in the same encephalic areas [4,5,12,31,32] suggests that the modulation of central as well as peripheral physiological processes may be regulated by the interaction between natriuretic factors and catecholamines. We have previously reported that ANF modulated noradrenergic neurotransmission in hypothalamus. Those results showed that ANF decreased noradrenergic neurotransmission since it enhanced neuronal NE uptake and NE endogenous content, whereas it diminished the synthesis and turnover as well as the output (spontaneous and evoked) of NE at the neuronal presynaptic level [20-24,33]. Furthermore, other authors have also reported ANF effects on catecholamines. Nakamaru and Inagami [34] showed that the electrically stimulated release of NE from postganglionic nerve endings was inhibited by ANF in a dose-dependent fashion. In addition, Debinski et al. [35] demonstrated that ANF inhibited stimulated catecholamine synthesis in the superior ganglia of the rat. Present results show that BNP and CNP display similar effects to those reported for ANF on NE uptake. In total NE uptake experiments, when equimolar concentrations (100 riM) of BNP and CNP were compared, the magnitude of CNP effect was always higher than that of BNP (91% vs. 69%). Both, BNP and CNP, increased only neuronal [3H]NE uptake (Co sensitive) and failed to modify nonneuronal NE uptake (HC sensitive). The increases for total NE uptake produced by 100 nM BNP and 1 nM CNP were very similar to those observed for the neuronal uptake of
the amine (BNP: 69.0% vs. 67.0% and CNP: 40.0% vs. 39.0%, respectively). Nevertheless, it is important to point out that the threshold concentration of CNP was 100-fold lower than that of BNP (1 nM vs. 100 nM, respectively). Threshold concentrations of both peptides caused a rapid increase on NE uptake at the first minute of the experimental period, which was sustained for 60 min. The neuronal uptake of NE allows the inactivation of catecholamines, being this mechanism mediated by an active transport system dependent on N a + / K + ATPase (affected by temperature and the extracellular Na ÷ concentration) [36]. Both, BNP and CNP may increase the availability of transporters already present or the synthesis of new transporters involved in the reuptake process of NE. A further possibility is that these peptides may alter the membrane potential and therefore the driving force on the coupled Na ÷. NE intracellular distribution was modified by CNP but not by BNP. CNP increased NE into the granular fraction and decreased the content of the cytosolic pool, an action similar to that of ANF [20,33]. The vesicular amine transport, dependent on Mg 2~ ATPase activity, increases H ÷ translocation from cytoplasm into the vesicle, thus reducing intragranular pH and inducing a voltage difference between cytoplasm and the vesicular compartment, which results in an enhancement of NE uptake into the vesicle [35]. Provided that CNP modified NE intracellular distribution increasing the granular fraction, it is possible to assume that this peptide may affect the vesicular amine transport in either a direct or an indirect way. Results also showed that BNP and CNP reduced spontaneous NE release being the percentual decreases for CNP (1, 10 and 100 riM, 24.0%, 27.0% and 33.0%, respectively) always higher than for BNP (100 nM, 16%). On the other hand, the lowest effective concentrations of CNP and BNP (1 pM and 1 nM, respectively) decreased KC1 evoked NE output (30.0% vs. 39.0%). However, when equimolar concentrations of both peptides (1 nM) were compared, the magnitude of the evoked NE release decrease induced by CNP was higher than that of BNP (45.0% vs. 39.0%). The effect of both peptides was more pronounced on KCl-induced NE release than on spontaneous NE output. It is important to point out that the threshold concentration for CNP, 100-times lower than that of BNP, to affect evoked NE release was 1 pM, which is within the physiological range. Neurotransmitter release, such as that of NE, is a calcium-dependent mechanism involving diverse cytoesqueleton proteins (synapsin I and II, fodrin, actin) which are intimately related to calcium-dependent mechanisms [37]. In addition, we have previously reported that ANF behaves as a calcium channel blocker [23]. According to present results it is likely that CNP and BNP could alter calcium metabolism and therefore interact with the NE exocytotic release. In addition, other mechanisms that may mediate natriuretic peptides effect on neurotransmission release include an inhibition of the nicotinic receptor
M.S. Vatta et al./Regulatory Peptides 65 (1996) 175-184
or a stimulation of the potassium channels. These mechanisms hyperpolarize the neurons, making them less excitable [38]. The difference in the relationship between the concentration and the effect achieved for BNP and CNP may result from the different distribution of the natriuretic factor receptors in the CNS. These results are closely related to the rank order of affinity for ANFR-B which is the receptor most widely distributed in the CNS [18]. Northern blotting experiments and cDNA cloning have demonstrated the expression of A N F R - A in the kidney, adrenal gland, ileum, placenta, pituitary and brain, and ANFR-B mainly in pituitary, brain and placenta [7,18,19]. A N F R - A responds to the stimulation by A N F and less to BNP and CNP (ANF > BNP > CNP), whereas ANFR-B displays a different hormonal selectivity to that of ANFR-A being CNP > ANF > BNP [18,19]. Several authors have demonstrated that BNP displayed inhibitory effects on angiotensin-II-induced dipsogenic behaviour, pressor response and vasopressin release as already reported for A N F [9,39,40]. Furthermore, angiotensin II and III modulates NE metabolism in several regions of the CNS [27,41]. BNP and CNP, as well as ANF, showed opposite actions to those of angiotensin II in the CNS. On this basis, the natriuretic factors could be a physiological system that antagonizes the rote of central nervous born angiotensins in the control of several mechanism such as control of blood arterial pressure, water and electrolyte homeostasis, thirst, and neuroendocrine secretions. In conclusion, present results suggest that BNP and CNP may modulate noradrenergic activity at the neuronal presynaptic level to affect physiological functions centrally mediated by catecholamines. The possible regulatory role for BNP and CNP is similar to that already reported for ANF. The data provide evidence to support the hypothesis that CNP may be the major natriuretic peptide that modulates NE metabolism in the CNS.
Acknowledgements This work was supported by grants of the Consejo Nacional de Investigaciones Cientfficas y TEcnicas (CONICET) (PID 3225) and Universidad de Buenos Aires (FA032), Repfiblica Argentina. We thank Dr. Edda Villamil from the Toxicology Department of the Faculty of Pharmacy and Biochemistry for the generous supply of cocaine chlorhydrate.
References [1] de Bold, A.J., Borenstein,H.B., Veress, A.T. and Sonnenberg,G.H., A rapid and potent natriuretic response to intravenous injection of atrial myocardialextract in rats, Life Sci., 28 (1981) 89-94. [2] Brenner, B.M., Ballermann, B.J., Gunning, M.E. and Zeidel M.L.,
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Diverse biologicalactions of atrial natriureticpeptide, Physiol. Rev., 70 (1990) 665-699. [3] Mangat, H. and de Bold, A.J., Stretch-induced atrial natriuretic factor release utilizes a rapidly depletingpool of newly synthesized hormone, Endocrinology,133 (1993) 1398-1403. [4] Itoh, H., Nakao, K., Saito, Y., Yamada, T., Shirikami, G., Mukoyama, M., Matsuo, H. and Imura, H., Radioimmunoassayfor brain natriuretic peptide (BNP) detection of BNP in canin brain, Biochem. Biophys. Res. Commun., 158 (1989) 120-128. [5] Saper, C.B., Hurley, K.M., Moga, M.M., Holmest, R.H., Adams, S.A., Leahy, K.M. and Needleman, P., Brain natriuretic peptides: differential localization of new family of neuropeptides, Neurosci. Lett., 96 (1989) 29-34. [6] Kamboyashi,Y., Nakao, K., Kimura, H., Kawabata, T., Nakamura, M., Inouye, K., Yoshira, N. and Imura, H., Biologicalcharacterization of human brain natriureticpeptide (BNP) and rat BNP: speciesspecific action of BNP, Biochem. Biophys. Res. Commun., 173 (1990) 599-605. [7] Rosenzweig, A. and Seidman, C.E., Atrial natriuretic factor and related peptide hormones,Annu. Rev. Biochem., 60 (199l) 229-255. [8] Nakao, K., Itoh, h., Kambayashi, Y., Hosoda, K., Saito, Y., Yamada, T., Mokoyama, M., Arai, H., Shirakami, G., Suga, S.T., Jougasaki, M., Ogawa, Y., Inouye, K. and lmura H., Rat brain natriuretic peptide. Isolation from heart and tissue distribution,Hypertension, 15 (1990) 774-778. [9] Itoh, H., Nakao, K., Yamada, T., Shirakami, G., Kangawa, K., Minamino,N., Matsuo, H. and lmura, H., Antidipsogenicaction of a novel peptide, 'brain natriureticpeptide', in rats, Eur. J. Pharmacol., 150 (1988) 193-196. [10] Jennings, D.B. and Flynn, T.G., Cardiovascularand renal effects of iso-r ANP, a second natriuretic peptide from rat atria, Can. J. Physiol. Pharmacol., 67 (1989) 1322-1329. [11] Sudoh, T., Minamino, N., Kangawa, K. and Matsuo, H., C-type natriuretic peptide (CNP): a new member of the natriureticpeptide family identified in porcine brain, Biochem. Biophys. Res. Commun., 168 (1990) 863-870. [12] Komatsu,Y., Nakao, K., Suga, S., Ogawa, Y., Mukoyama,M., Arai, H., Shirakami, G., Hosoda, K., Nakagama, O., Hama, N., Kishimoto, 1. and Imura, H., C-Type natriureticpeptide (CNP) in rats and humans, Endocrinology,129 (1991) 1104-1106. [13] Stingo, A.J., Clavell, A.L., Aarhus, L. and Bumett Jr, J.C, Cardiovascular and renal actions of C-type natriuretic peptide, Am. J. Physiol. (Heart Circ. Physiol. 31), 262 (1992) H308-H312. [14] Clavell, A.L., Stingo,A.J., Wei, Ch., Heublein,D.M. and BurnettJr, J.C., C-type natriuretic peptide: a selective cardiovascularpeptide, Am. J. Physiol. (Regul. Integr. Comp. Physiol., 33), 264 (1993) R290-R295. [15] Stingo, A.J., Clavell, A.L., Heublein, D.M., Wei, Ch., Pittelkow, M.R. and Barnett Jr, J.C., Presence of C-type natriureticpeptide in cultured human endothelialcells and plasma, Am. J. Physiol. (Heart Circ. Physiol., 32), 263 (1992) HI318-HI321. [16] Heublein, D.M., Clavell, A.L., Stingo, A.J., Lerman, A., Wold, L. and Burnett Jr, J.C., C-type natriureticpeptide immunoreactivityin human breast vascular endothelial cells, Peptides, 13 (1992) 10171019. [17] Wei, Ch., Aarhus, L.L., Miller, V.M. and BurnettJr., J.C., Action of C-type natriureticpeptide in isolated canine arteries and veins, Am. J. Physiol. (Heart Circ. Physiol., 33), 264 (1993) H71-H73. [18] Koller, K.J., Lowe, D.G., Bennett, G.L., Minamino, N., Kangawa. K., Matsuo, H. and Goeddel, D.V., Selective activation of the B natriuretic peptide receptor by C-type natriuretic peptide (CNP), Science, 252 (1991) 120-123. [19] Suga, S., Nakao, K., Hosoda, K., Mukoyama, M., Ogawa, Y., Shirakami, G., Arai, H., Saito, Y., Kambayashi,Y., Iuouye, K. and Imura, H., Receptor selectivity of natriuretic peptide family, atrial natriuretic peptide, brain natriuretic peptide and C-type natriuretic peptide, Endocrinology,130 (1992) 229-239.
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[20] Fernandez, B.E., Vatta, M.S., Bianciotti, L.G. and Mart~nez Seeber. A., Atrial natriuretic peptide: Effects on uptake and intracellular distribution of norepinephrine in hypothalamus, Pharmacology (Life Sci. Adv.), 9 (1990) 525-533. [21] Vatta, M.S., Bianciotti, L.G., Papuchado, M.L., Locatelli, A.S. and Fernandez, B.E., Effects of atrial natriuretic peptide and angiotensin III on the uptake and intracellular distribution of norepinephrine in medulla oblongata of the rat, Comp. Biochem. Physiol., 99C (1991) 293-297. [22] Vatta, M.S., Travaglianti, M., Bianciotti, L., Coil, C., Perazzo, J. and Fernandez, B.E., Atrial natriuretic factor effects on norepinephrine uptake in discrete telencephalic and diencephalic nuclei of the rat, Brain Res., 646 (1994) 324-326. [23] Vatta, M.S., Papouchado, M.L., Locatelli, A.S., Bianciotti, L.G. and Fernfindez, B.E., Effects of atrial natriuretic factor on norepinephrine release in the rat hypothalamus, Regul. Pept., 41 (1992) 171-181. [24] Vatta, M.S., Papuchado, M.L., Bianciotti, L.G. and Fernfindez, B.E., Atrial natriuretic factor inhibits noradrenaline release in presence of angiotensin II and III in the rat hypothalamus, Comp. Biochem. Physiol., 106C (1993) 545-548. [25] Kimelberg, H.K. and Goderie, S.K., Effect of ascorbate on Na---independent and Na÷-dependent uptake of [3H]norepinephrine by rat primary astrocyte cultures from neonatal rat cerebral cortex, Brain Res., 603 (1993) 41-44. [26] Glowinski, J. and Axelrod. J., Effects of drugs on the disposition of H-norepmephnne in the rat brain, Pharmacol. Rev., 18 (1966) 775-785. [27] Sumners, C. and Raizada, M.K., Angiotensin II stimulates norepinephrine uptake in hypothalamus brain-stem neuronal cultures, Am. J. Physiol. (Cell Physiol., 19), 250 (1986) C236-C244. [28] Palaic, D. and Khairallah, P.A., Effects of angiotensin on uptake and release of norepinephrine by brain, B iochem. Pharmacol., 16 (1967) 2291-2298. [29] Wallenstein, S., Zucker, C. and Fleiss, J.L., Some statistical methods useful in circulation research, Circ. Res., 47 (1980) 1-9. [30] Marley, E. and Stephenson, J.D., Central actions of Catecholamines. In H. Blaschko and E. Muscholl (Eds.), Handbook of Experimental Pharmacology XXXIII - Catecholamines. Springer-Verlag, Berlin, 1972, Chapter 12, pp. 463-537. 3
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[31] Saavedra, J.M., Grobecker, H. and Axelrod, J., Changes in central catecholaminergic neurons in spontaneously (genetic) hypertensive rat, Circ. Res., 42 (1978) 529-534. [32] Skofitsch, G. and Jacobowitz, D.M., Atrial natriuretic peptide in the central nervous system of the rat, Cell Mol. Neurobiol., 8 (1988) 339-391. [33] Fernandez, B.E., Dom~nguez, A.E., Vatta, M.S., Mrndez, M.A., Bianciotti, L.G. and Feldstein, C.A., Atrial natriuretic peptide modulates norepinephrine metabolism, J. Hypertens. (Abstr.), 8 (1990) 544. [34] Nakamaru, M. and Inagami, T., Atrial natriuretic factor inhibits norepinephrine release evoked by sympathetic nerve stimulation in isolated perfused rat mesenteric arteries, Eur. J. Pharmacol., 123 (1986) 459-461. [35] Debinski, W., Kuche, O., Nguyen, T.B., Cantin, M. and Genest, J., Atrial natriuretic factor partially inhibits the stimulated catecholamine syntesis in superior cervical ganglia of the rat, Neurosci. Lett., 77 (1987) 92-96. [36] Philippu, A. and Matthaei, H., Transport and storage of catecholamines in vesicles. In U. Trendelenburg and N. Weiner (Eds.), Handbook of Experimental Pharmacology - Catecholamines I. Springer-Verlag, Berlin, 1988, Vol. 90/I, Chapter 1, pp. 1-42. [37] Trifarr, J.M. and Vitale, M.L., Cytoskeletou dynamics during neurotransmitter release, Trends Neurosci., 16 (1993) 466-472. [38] Anand-Srivastava, M.B. and Trachte, G.J., Atrial natriuretic factor receptors and signal transduction mechanisms, Pharmacol. Rev., 45 (1993) 455-497. [39] Shirikami, G., Nakao, K., Yamada, T., Itoh, H., Mori, K., Kangawa, K., Minamino, N., Matsuo, H. and Imura, H., Inhibitory effect of brain natriuretic peptide on central angiotensin II-stimulated pressor response in conscious rats, Neurosci. Lett., 91 (1988) 77-83. [40] Yamashita, H., and Kannan, h., Inhibition of hypothalamic neurons by the atrial natriuretic peptide family, News Physiol. Sci., 7 (1992) 75-79. [41] Papouchado, M.L., Vatta, M.S., Escalada, A., Bianciotti, L.G. and Fernandez, B.E., Angiotensin III modulates noradrenaline uptake and release in the rat hypothalamus, J. Auton. Pharmacol., 15 (1995) 1-8.
PEPTIDES
ELSEVIER
Regulatory Peptides 65 (1996) 175-184
B and C types natriuretic peptides modulate norepinephrine uptake and release in the rat hypothalamus M.S. Vatta *, M. Presas, L.G. Bianciotti, V. Zarrabeitia, B.E. Fernfindez Cdtedras de Fisiopatologia and Fisiologfa, Programa de Sistemas Vasodepresores - Consejo Naeional de lnvestigaciones Cientfficas y Tdcnicas (PROS1VAD-CONICET), Facultad de Farmacia y Bioqufmica, Universidad de Buenos Aires, Buenos Aires, Argentina Received 8 February 1996; revised 11 May 1996; accepted 20 May 1996
Abstract
We previously reported that atrial natriuretic factor (ANF) regulates catecholamine metabolism in the central nervous system. ANF, B and C types natriuretic peptides (BNP and CNP) also play a regulatory role in body fluid homeostasis, cardiovascular activity and hormonal and neuro-hormonal secretions. The aim of the present work was to investigate BNP and CNP effects on the uptake and release of norepinephrine (NE) in rat hypothalamic slices incubated in vitro. Results showed that BNP (100 riM) and CNP (1, 10 and 100 nM) enhanced total and neuronal [3H]NE uptake but did not modify non-neuronal uptake. BNP (100 nM) and CNP (1 nM) caused a rapid increase in NE uptake (1 rain), which was sustained for 60 min. BNP (100 nM) did not modify the intracellular distribution of NE; however, 1 nM CNP increased the granular store and decreased the cytosolic pool of NE. BNP (100 nM) and CNP (1, 10 and 100 riM), diminished spontaneous NE release. In addition, BNP (1, 10, 100 nM) and CNP (1, 10 and 100 pM, as well as 1, 10 and 100 nM) reduced NE output induced by 25 mM KC1. These results suggest that BNP and CNP may be involved in the regulation of several central as well as peripheral physiological functions through the modulation of noradrenergic neurotransmission at the presynaptic neuronal level. Present results provide evidence to consider CNP as the brain natriuretic peptide since physiological concentrations of this peptide (pM) diminished NE evoked release. Keywords: Neuronal norepinephrine uptake; Non-neuronal norepinephrine uptake; Norepinephrine intracellular distribution; Central nervous system; Atrial natriuretic factor
1. Introduction
Atrial natriuretic factor or A type natriuretic factor (ANF) originally discovered by de Bold et al. [1], is synthesized, stored and released by mammalian atrial cardiocytes in response to atrial stretch [2,3]. Several reports have demonstrated that, in fact, a family of natriuretic peptides exists: ANF, B type natriuretic peptide (BNP) and C type natriuretic peptide (CNP). Although BNP was initially identified in porcine brain, this peptide is more abundant in cardiac atriae and ventricles than in the central nervous system (CNS) [4-6]. Its amino acid sequence and biological actions are very similar to those of ANF. In the human and pig, a 32-amino-acid peptide is the circulating form of BNP [7], while in rodents
* Corresponding author. C~tedra de Fisiopatologla, Facultad de Farmacia y Bioqulmica, Universidad de Buenos Aires. Jun~n 956 - 5to piso, (1113) Buenos Aires, Argentina.
the 45-amino-acid sequence is the mature form of BNP [8]. Circulating BNP may play a role in regulating water and electrolyte balance and cardiovascular homeostasis [6,7,9,10]. The newest member of the natriuretic family is CNP, which is a 22-amino-acid peptide that has been identified in the CNS, kidney and intestine of pig, human and rat [11,12]. Burnett and co-workers have demonstrated that the intravenous administration of CNP causes a marked decrease in blood arterial pressure, cardiac filling pressure and cardiac output without natriuresis [13,14]. CNP circulates in low picomolar concentrations and is a potently vasoactive in vivo, suggesting a potential role in the regulation of vascular tone [14,15]. CNP immunoreactivity is present in human endothelial cells and it could be an important component of an autocrine endothelium-derived vasomodulatory system [ 13,16,17]. Two types of natriuretic factor receptor have been identified in target tissues: two different guanylate cyclase linked receptors that appear to mediate most of the biologi-
0167-0115/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PI1 S01 6 7 - 0 1 1 5 ( 9 6 ) 0 0 0 9 0 - 0
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cal effects (ANFR-A and ANFR-B) and the non-guanylate coupled receptor or clearance receptor (ANFR-C) which appears to regulate the natriuretic factors circulating levels [2,7]. Nevertheless it has been demonstrated that in several tissues the latter may be biologically active. It does not signal through activation of guanylyl cyclase, but may function through intermediate G-protein to inhibit adenylate cyclase or activate phosphoinositide pathway [7,18,19]. Both ANFR-A and ANFR-B are widely distributed in the CNS and other peripheral tissues [7,18,19]. We have previously reported that, in the CNS of the rat, ANF increases neuronal norepinephrine (NE) uptake and NE content into the granular fraction [20-22]. In addition, it diminishes spontaneous as well as evoked NE release [23,24] and NE turnover (unpublished data). In view of ANF effects on noradrenergic neurotransmission, the presence of BNP and CNP immunoreactivity in 45-
several encephalic regions such as hypothalamus and ANF-A and ANF-B receptors distribution in the CNS, we considered it of interest to study BNP and CNP effects on: (a) total [3H]NE uptake; (b) time course of [3H]NE uptake; (c) neuronal and non-neuronal [3H]NE uptake; (d) intracellular distribution of [3H]NE; and (e) spontaneous and evoked neuronal release of [3 H]NE.
2. Materials and methods 2.1. Animals
Wistar strain male rats weighing between 250-300 g (From the Pathophysiology Department, Faculty of Pharmacy & Biochemistry, UBA, Buenos Aires, Argentina) were used in all experiments. The rats were housed in steel
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M.S. Vatta et al./Regulatot T Peptides 65 (1996) 175-184
2.3. Experimental procedures
cages and maintained at a temperature between 22-24°C in a controlled room with a 12 h light/dark cycle (light from 7:00 to 19:00 h). Animals were allowed free access to tap water and food (commercial rodents Purina chow).
All experiments were carried out in vitro.
2.2. Drugs and solutions The following drugs were used in the experiments: NE-HC1 DL [7-3H(N)] (New England Nuclear, Boston, MA, USA; of 15 C i / m m o l of specific activity; rat BNF-32 (Sigma, St. Louis, MO, USA); CNF (1-22) (Peptide Institute, Osaka, Japan); hydrocortisone (HC) and pargyline (PRG) (Sigma) and cocaine chloridrate (Co) (supplied by the Toxicology Department, Faculty of Pharmacy & Biochemistry, UBA). Incubation medium: Standard Krebs bicarbonate solution of the following composition (in mM): NaC1 118; KC1 4.7; MgC12 1.2; NaH2PO 4 1.0; CaC12 2.5; EDTA-Na 0.004; dextrose 11.1; NaHCO 3 25.0; ascorbic acid 0.11.
2.3.1. Total [ SH]NE uptake Animals were decapitated between 10:00 and 12:00 a.m. to avoid circadian changes. Brains were quickly removed and hypothalami were immediately dissected, cooled and weighed. Slices were cut and then transferred into a glass tube with a mesh of nylon fitted at the bottom to allow free interchange with the medium. Slices were placed in a Dubnoff incubator and pre-incubated for 15 min at 37°C (pH 7.4) and bubbled with carbogen (95% O2/5% CO 2) under continued shaking with 2 ml of Krebs bicarbonate solution. Monoamine oxidase (MAO) activity was inhibited by the addition of PRG (100 mM) for 30 rain. NE stores were labelled during the 30 min incubation period [25] with 1.25 mCi/ml of [3H]]NE in the absence (control group) or in the presence of BNP (1, 10 and 100
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M.S. Vatta et al. / Regulatory Peptides 65 (1996) 175-184
178
nM) or CNP (10 and 100 pM; 1, 10 and 100 nM) (experimental groups). During this period MAO activity was also inhibited with 100 mM PRG. Slices were washed for 5 min with 2 ml of Krebs bicarbonate solution and were then homogenized with 2.5 ml of 10% trichloroacetic acid and centrifuged at 27000 × g and 4°C for 15 min (Sorvall Superspeed RC2-B). Tritium activity was determined in supernatants by usual scintillation counting methods (Packard-PRIAS 240CL/D).
scribed for total [3H]NE uptake determination. NE uptake was determined at 1, 5, 10, 30 and 60 min. The following groups were studied: (a) control slices; (b) slices incubated with 100 nM BNP; and (c) slices incubated with 1 nM CNP.
2.3.3. Neuronal and non-neuronal [3H]NE uptake Several authors have reported that neuronal NE uptake did not differ in reserpined tissues when compared with non-reserpined tissues [26,27]. Therefore, experiments were carried out in non-reserpined tissues. Hypothalamic slices were submitted to the same procedures as described for total NE uptake determination, except that neuronal and non-neuronal [3H]NE uptake were inhibited by 10 mM Co
2.3.2. Time-course of [~H]NE uptake Threshold concentrations of BNP and CNP were chosen according to total [3H]NE uptake study. Tissues were submitted to the same experimental procedures as de35-
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Fig. 3. Effects of BNP (a) and CNP (b) on neuronal and non-neuronal [3H]NE uptake. CO, cocaine chlorhydrate; HC, hydrocortisone. Data are shown as mean _+ SEM (n = 6-8). * P < 0.05 compared with control; * * P < 0.05 compared with HC.
M.S. Vatta et al. / Regulatory Peptides 65 (1996) 175-184
and 100 mM HC, respectively. These inhibitions were carried out 30 min before and during the incubation period. The following groups were studied: (a) control and incubated with: (b) HC; (c) HC + 100 nM BNP; (d) HC + 1 nM BNP; (e) Co; (f) Co + 100 nM BNP; and (g) Co + 1 nM CNP.
2.3.4. Intracellular distribution of [YH]NE Hypothalamic tissues were submitted to the same experimental procedures as described for total NE uptake determination except that after the washing period, 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 (supernatant) and the granular (pellet) fractions [28]. Both pools were deproteinized with 10% trichloroacetic acid and centrifuged at 27000 × g for 15 min. Tritium activity was QO
179
measured in the supernatants of the following groups: (a) control and incubated with: (b) 100 nM BNP and (c) 1 nM CNP.
2.3.5. Neuronal [3H]NE release Hypothalamic slices were pre-incubated for 15 min at 37°C (pH 7.4) and bubbled with carbogen (95% 0 2 / 5 % CO2) under continuous shaking with 2 ml of Krebs bicarbonate solution. Then, MAO activity and extraneuronal uptake of NE were inhibited by the addition of 100 mM PRG and 100 mM HC, respectively, 30 min before and during the incubating period (30 min). In this last period, tissues were also labelled with [3H]NE. To avoid extracellular release and to obtain the equillibrium of the basal neuronal release, hypothalami were washed for 30 min with 25 ml of modified Krebs solution (in six consecutive washing periods of 5 min). The tissues were then incu-
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bated for 10 min and two consecutive samples of the medium were collected every 5 min. The first sample corresponded to the basal release period and the second sample belonged to the experimental period. Hypothalami were divided into groups for: (a) the determination of spontaneous neuronal [3H]NE release in control group and groups treated with BNP (1, 10, 100 nM) and CNP (10, 100 pM and 1, 10, 100 nM), and (b) the determination of evoked neuronal release of [3H]NE in control group and groups treated with 25 mM KC1 or 25 mM KC1 plus BNP (10, 100 pM and 1, 10, 100 nM) or CNP (10 and 100 fM; 1, 10 and 100 pM; 1, 10 and 100 nM). The volumes of all the samples were reduced to 0.2 ml in a vacuum stove (National Appliance, Model 5831) and 3H activity was measured by usual scintillation counting methods. 2.4. Analysis of results
NE uptake is expressed as d p m / g of the fresh tissues _+ SEM. Intracellular distribution of NE is expressed as the 1-
percentage + SEM 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 _ SEM. One-way analysis of variance (ANOVA) and the t-test modified by Bonferroni were used for statistical analysis [29]. P values of 0.05 or less were considered statistically significant.
3. Results 3.1. Total [3H]NE uptake
Concentration-response studies were carried-out using increasing concentrations of BNP and CNP in order to determine if these peptides modified total [3H]NE uptake. Fig. la and lb illustrate the threshold and subthreshold concentrations of BNP and CNP on NE uptake. Results showed that 1 and 10 nM BNP did not modify total [3H]NE uptake, whereas 100 nM BNP increased the amine
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Fig. 5. Effects of BNP (a) and CNP (b) on spontaneous [3H]NE release. Open column: control; down-hatchedcolumn: BNP; up-hatched column: CNP. Data are shown as mean + SEM (n = 6-8). * P < 0.05 comparedwith control.
M.S. Vatta et al. / Regulator)' Peptides 65 (1996) 175-184 uptake (Fig. la). On the other hand, l0 and 100 pM CNP failed to modify total NE uptake; however, 1, 10 and 100 nM C N P increased total [3H]NE uptake in a concentration-dependent fashion (Fig. lb).
181
3.3. Neuronal and non-neuronal [ 3H]NE uptake The aim of this study was determine if the increase observed in total NE uptake was due to an increase in either neuronal or non-neuronal [3H]NE uptake. When neuronal [3H]NE uptake was inhibited by 10 mM Co, the residual non-neuronal uptake was not modified by threshold concentrations of either BNP (100 nM) or CNP (1 riM). Nevertheless, when non-neuronal [3H]NE uptake was inhibited by 100 mM HC, the remaining neuronal NE uptake was increased by threshold concentrations of both BNP and CNP (Fig. 3a and b).
3.2. Time-course of [3H]NE uptake Fig. 2a and b show the modifications on NE uptake along the experimental period of 60 min. Threshold concentrations of both BNP (100 nM) and CNP (1 nM) increased total [3H]NE uptake in the first minute of the experimental period as compared to control time course. The curve of BNP on total NE uptake reached its highest level at 30 min and remained significantly higher than the control curve up to the end of the study (60 min) (Fig. 2a). On the other hand, CNP time-course showed a progressive increase in [3H]NE uptake (from 1 min to 60 min) reaching the maximum value at the end of the experimental period (Fig. 2b).
3.4. Intracellular distribution of [ 3H]NE Results showed that 100 nM BNP did not alter the intracellular distribution of NE (Fig. 4a). However, a threshold concentration of CNP (1 nM) diminished NE
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Fig. 6. Effects of BNP (a) and CNP (b) on [3H]NE release evoked by 25 mM KCI. Open column: control; solid column: KC1; dora-hatched column: KCI + BNP; up-hatched column: KCI + CNP. Data are shown as mean _+SEM (n = 5-9). * P < 0.05 compared with control; * * P < 0.05 compared with KC1.
182
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cytosolic pool content and increased NE vesicular pool content (Fig. 4B). 3.5. N e u r o n a l [ 3H ] N E release
Both BNP and CNP modified spontaneous, as well as evoked neuronal release of NE. 1 and 10 nM BNP, as well as 10 and 100 pM CNP did not modify spontaneous neuronal output of the amine. However, 100 nM BNP and 1, 10, 100 nM CNP diminished spontaneous neuronal NE secretion, in a concentration dependent fashion, as compared with the control group (Fig. 5a and b). On the other hand, Fig. 6a and b show that 1, 10, 100 nM BNP and 1, 10 and 100 pM as well as 1, 10 and 100 nM CNP decreased [3H]NE secretion evoked by a high potassium despolarizing solution (25 mM KC1). Conversely, 10 and 100 pM BNP as well as 10 and 100 fM CNP did not alter the evoked release of NE.
4. Discussion Hypothalamic and brain catecholamines are involved in the regulation of several biological actions such as cardiovascular activity, water and electrolyte balance, endocrine and neuroendocrine synthesis and secretion, thirst and apatite, etc. [30]. The existence of immunoreactive natriuretic factors and catecholamines in the same encephalic areas [4,5,12,31,32] suggests that the modulation of central as well as peripheral physiological processes may be regulated by the interaction between natriuretic factors and catecholamines. We have previously reported that ANF modulated noradrenergic neurotransmission in hypothalamus. Those results showed that ANF decreased noradrenergic neurotransmission since it enhanced neuronal NE uptake and NE endogenous content, whereas it diminished the synthesis and turnover as well as the output (spontaneous and evoked) of NE at the neuronal presynaptic level [20-24,33]. Furthermore, other authors have also reported ANF effects on catecholamines. Nakamaru and Inagami [34] showed that the electrically stimulated release of NE from postganglionic nerve endings was inhibited by ANF in a dose-dependent fashion. In addition, Debinski et al. [35] demonstrated that ANF inhibited stimulated catecholamine synthesis in the superior ganglia of the rat. Present results show that BNP and CNP display similar effects to those reported for ANF on NE uptake. In total NE uptake experiments, when equimolar concentrations (100 riM) of BNP and CNP were compared, the magnitude of CNP effect was always higher than that of BNP (91% vs. 69%). Both, BNP and CNP, increased only neuronal [3H]NE uptake (Co sensitive) and failed to modify nonneuronal NE uptake (HC sensitive). The increases for total NE uptake produced by 100 nM BNP and 1 nM CNP were very similar to those observed for the neuronal uptake of
the amine (BNP: 69.0% vs. 67.0% and CNP: 40.0% vs. 39.0%, respectively). Nevertheless, it is important to point out that the threshold concentration of CNP was 100-fold lower than that of BNP (1 nM vs. 100 nM, respectively). Threshold concentrations of both peptides caused a rapid increase on NE uptake at the first minute of the experimental period, which was sustained for 60 min. The neuronal uptake of NE allows the inactivation of catecholamines, being this mechanism mediated by an active transport system dependent on N a + / K + ATPase (affected by temperature and the extracellular Na ÷ concentration) [36]. Both, BNP and CNP may increase the availability of transporters already present or the synthesis of new transporters involved in the reuptake process of NE. A further possibility is that these peptides may alter the membrane potential and therefore the driving force on the coupled Na ÷. NE intracellular distribution was modified by CNP but not by BNP. CNP increased NE into the granular fraction and decreased the content of the cytosolic pool, an action similar to that of ANF [20,33]. The vesicular amine transport, dependent on Mg 2~ ATPase activity, increases H ÷ translocation from cytoplasm into the vesicle, thus reducing intragranular pH and inducing a voltage difference between cytoplasm and the vesicular compartment, which results in an enhancement of NE uptake into the vesicle [35]. Provided that CNP modified NE intracellular distribution increasing the granular fraction, it is possible to assume that this peptide may affect the vesicular amine transport in either a direct or an indirect way. Results also showed that BNP and CNP reduced spontaneous NE release being the percentual decreases for CNP (1, 10 and 100 riM, 24.0%, 27.0% and 33.0%, respectively) always higher than for BNP (100 nM, 16%). On the other hand, the lowest effective concentrations of CNP and BNP (1 pM and 1 nM, respectively) decreased KC1 evoked NE output (30.0% vs. 39.0%). However, when equimolar concentrations of both peptides (1 nM) were compared, the magnitude of the evoked NE release decrease induced by CNP was higher than that of BNP (45.0% vs. 39.0%). The effect of both peptides was more pronounced on KCl-induced NE release than on spontaneous NE output. It is important to point out that the threshold concentration for CNP, 100-times lower than that of BNP, to affect evoked NE release was 1 pM, which is within the physiological range. Neurotransmitter release, such as that of NE, is a calcium-dependent mechanism involving diverse cytoesqueleton proteins (synapsin I and II, fodrin, actin) which are intimately related to calcium-dependent mechanisms [37]. In addition, we have previously reported that ANF behaves as a calcium channel blocker [23]. According to present results it is likely that CNP and BNP could alter calcium metabolism and therefore interact with the NE exocytotic release. In addition, other mechanisms that may mediate natriuretic peptides effect on neurotransmission release include an inhibition of the nicotinic receptor
M.S. Vatta et al./Regulatory Peptides 65 (1996) 175-184
or a stimulation of the potassium channels. These mechanisms hyperpolarize the neurons, making them less excitable [38]. The difference in the relationship between the concentration and the effect achieved for BNP and CNP may result from the different distribution of the natriuretic factor receptors in the CNS. These results are closely related to the rank order of affinity for ANFR-B which is the receptor most widely distributed in the CNS [18]. Northern blotting experiments and cDNA cloning have demonstrated the expression of A N F R - A in the kidney, adrenal gland, ileum, placenta, pituitary and brain, and ANFR-B mainly in pituitary, brain and placenta [7,18,19]. A N F R - A responds to the stimulation by A N F and less to BNP and CNP (ANF > BNP > CNP), whereas ANFR-B displays a different hormonal selectivity to that of ANFR-A being CNP > ANF > BNP [18,19]. Several authors have demonstrated that BNP displayed inhibitory effects on angiotensin-II-induced dipsogenic behaviour, pressor response and vasopressin release as already reported for A N F [9,39,40]. Furthermore, angiotensin II and III modulates NE metabolism in several regions of the CNS [27,41]. BNP and CNP, as well as ANF, showed opposite actions to those of angiotensin II in the CNS. On this basis, the natriuretic factors could be a physiological system that antagonizes the rote of central nervous born angiotensins in the control of several mechanism such as control of blood arterial pressure, water and electrolyte homeostasis, thirst, and neuroendocrine secretions. In conclusion, present results suggest that BNP and CNP may modulate noradrenergic activity at the neuronal presynaptic level to affect physiological functions centrally mediated by catecholamines. The possible regulatory role for BNP and CNP is similar to that already reported for ANF. The data provide evidence to support the hypothesis that CNP may be the major natriuretic peptide that modulates NE metabolism in the CNS.
Acknowledgements This work was supported by grants of the Consejo Nacional de Investigaciones Cientfficas y TEcnicas (CONICET) (PID 3225) and Universidad de Buenos Aires (FA032), Repfiblica Argentina. We thank Dr. Edda Villamil from the Toxicology Department of the Faculty of Pharmacy and Biochemistry for the generous supply of cocaine chlorhydrate.
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