Brain natriuretic peptide-mediated changes in the extracellular neurotransmitter turnover in the rostral ventrolateral medulla

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Neuroscience Vol. 84, No. 1, pp. 255–262, 1998 Copyright ? 1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(97)00495-8

BRAIN NATRIURETIC PEPTIDE-MEDIATED CHANGES IN THE EXTRACELLULAR NEUROTRANSMITTER TURNOVER IN THE ROSTRAL VENTROLATERAL MEDULLA B. R. DEV,*‡ M. NANDAKUMARAN,† L. PHILIP* and S. J. JOHN* Departments of *Physiology, and †Obstetrics and Gynecology, Health Sciences Center—Faculty of Medicine, Kuwait University, P.O. Box 24923, Safat, Kuwait 13110 Abstract––Changes in the rostral ventrolateral medullary neurotransmitter levels and associated cardiovascular functions in response to local administration of brain natriuretic peptide were investigated in urethane-anesthetized Sprague–Dawley rats. Unilateral injections of various doses of brain natriuretic peptide into the rostral ventrolateral medulla region led to significant reductions in both blood pressure and heart rate. To identify the changes occurring in the extracellular neurochemical profile, brain natriuretic peptide was perfused at the rate of 1.5 µl/min for a period of 1 h through a microdialysis probe implanted stereotaxically into the rostral ventrolateral medulla area and the dialysate was assayed every 15 min for both catechols and indoleamine. Both norepinephrine and epinephrine concentrations were significantly reduced. Dihydroxyphenylacetic acid concentration showed no significant change in response to brain natriuretic peptide perfusion. On the other hand, serotonin turnover estimated by the measurement of its metabolite (5-hydroxyindoleacetic acid) concentration increased during the perfusion of brain natriuretic peptide. Blood pressure and heart rate also showed significant reduction during the perfusion of brain natriuretic peptide. These results suggest that brain natriuretic peptide may be relevant in the central regulation of cardiovascular functions by modulating monoamine neurotransmitters. ? 1998 IBRO. Published by Elsevier Science Ltd. Key words: blood pressure, dihydroxyphenylacetic acid, epinephrine, 5-hydroxyindoleacetic acid, microdialysis, norepinephrine.

Brain natriuretic peptide (BNP) was isolated and purified as a 26-amino acid residue from porcine brain.42 Subsequently, another BNP with a 32-amino acid (BNP-32) sequence was isolated from porcine brain and was found to have physiological activities similar to those of BNP-26.43 BNP has also been isolated from the atrial tissues of rat, pig and humans.1,2,17,22,31 Reports involving the regional distribution of both atrial natriuretic peptide (ANP) and BNP in rat,40 canine19 and porcine45 brain have demonstrated differential distributions of these two peptides. The immunoreactive BNP in porcine whole brain was found to be much greater than that of immunoreactive ANP,45 suggesting the importance of BNP and its specific role in the CNS. Plasma BNP concentration was found to be higher in people with cardiovascular dysfunctions, such as ‡To whom correspondence should be addressed. Abbreviations: ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; DOPAC, 3,4-dihydroxyphenylacetic acid; EDTA, ethylenediaminetetra-acetate; 5-HIAA, 5-hydroxyindoleacetic acid; HPLC, high-performance liquid chromatography; NTS, nucleus of the solitary tract; PBS, phosphate-buffered saline; RVLM, rostral ventrolateral medulla; SHR, spontaneously hypertensive rat; WKY, Wistar–Kyoto normotensive rat.

congestive heart failure, essential hypertension, pregnancy-induced hypertension, acute myocardial infarction and other cardiac disorders.4,9,10,20,35,48 An increased plasma concentration of BNP was found during the hypertensive phase in spontaneously hypertensive rats (SHRs),27 in line with the observed positive correlation between human hypertension and BNP. Intravenous injection of BNP into anesthetized deoxycorticosterone acetate hypertensive rats24 and SHRs25 produced a rapid and significant natriuresis and hypotension. Local injection of various doses of BNP into the nucleus of the solitary tract (NTS) led to pronounced hypotension and bradycardia only in SHRs, not in Wistar–Kyoto normotensive (WKY) or Sprague–Dawley rats.13 Also, intracerebroventricular (i.c.v.) injection of BNP into conscious, unrestrained Wistar rats was shown to have no effect on basal blood pressure and heart rate.41 The ventrolateral medulla of the brainstem and its influence on the tonic control of the vasomotor tone has been well documented.8,12,34,37 The rostral ventrolateral medulla (RVLM), also known as the C1 region, receives innervation from the neurons of the NTS,39 which in turn receives peripheral baroreflex information arising from the carotid sinus and aortic

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baroreceptors.11,28 Direct projections of the adrenergic neurons of the RVLM to the intermediolateral and intermediomedial cell columns of the spinal cord and their importance in the regulation of the sympathetic outflow have been demonstrated.36,38 We have previously reported significant changes in the extracellular catecholamine and indoleamine levels in the RVLM during phenylephrine-induced hypertension and nitroprusside-induced hypotension.5,14 Though there are reports pertaining to the centrally mediated BNP effect on cardiovascular functions, there is no known report of neurotransmitter changes brought about by the perfusion of BNP into vasomotor areas, such as the RVLM. Hence, this study was carried out to define the neuromodulatory role of BNP and associated cardiovascular changes. EXPERIMENTAL PROCEDURES

Animal care Male Sprague–Dawley rats (Charles River Kingston, Stone Ridge, NY, U.S.A.), weighing 300–350 g, were used in this present study. These rats were housed in transparent polycarbonate cages with stainless steel top measuring 60#38#20 cm3 in groups of two to three animals per cage in the animal resources center situated at the Faculty of Medicine, Kuwait University under a 14-h light and 10-h dark cycle. The animal room temperature was maintained between 21 and 23)C, with a relative humidity of 50&10%. These rats had free access to food (rat chow from Pillsbury, Birmingham, U.K.) and water till the day of the experiment. Animals used in this study were obtained, maintained and used in accordance with the Animal Welfare Act and the ‘‘Guide for the Care and Use of Laboratory Animals’’ prepared by the Institute of Laboratory Animal Resources, National Research Council, U.S.A. General surgical procedures On the day of the experiment, the rats were anesthetized with intraperitoneal injection of urethane (1.5 g/kg body weight) and had the femoral artery cannulated (PE-50 polyethylene tubing) to measure the blood pressure and heart rate. These rats were artificially ventilated using a rodent respirator (Harvard Apparatus, Millis, MA, U.S.A., model 681) with room air through a tracheal cannula of polyethylene PE-240 tubing. Body temperature was maintained at 37&0.5)C with an electrically operated heating pad. A 32-gauge stainless steel tube attached to a PE-10 polyethylene tubing was lowered into the RVLM area using the coordinates of Paxinos and Watson.33 A Hamilton microliter syringe was used to inject the peptide through the 32-gauge cannula. The upper incisor bar of the stereotaxic frame (Stoelting, Wood Dale, IL, U.S.A.) was kept 3.4 mm below horizontal zero. The interaural line was used as the rostral–caudal zero point. Coordinates used were: A "3.8 mm, L +2.0 mm and V "10.2 mm from the skull surface. Local microinjection studies Both blood pressure and heart rate were allowed to stabilize for at least 2 h following unilateral implantation of the microinjection cannula into the RVLM. Blood pressure and heart rate were monitored throughout the experiment using a physiological transducer (SensoNor 840, Horten, Norway) attached to a Lectromed-MultiTrace 2 physiograph (Lectromed Ltd, Jersey, Channel Islands). Baseline values of both blood pressure and heart rate represent the

values recorded at the end of 2 h. Phosphate-buffered saline (PBS; pH 7.4) was used as a vehicle and all the dilutions of rat BNP-32 (mol. wt 3453; Sigma, St Louis, MO, U.S.A.) were done with the PBS (pH 7.4). These rats were injected with 2, 4, 20 and 40 pmol of BNP in a volume of 100 nl, with a 1-h interval between successive injections. Measurements of both blood pressure and heart rate were carried out throughout the experiment. At the end of each experiment, 1% aqueous Cresyl Fast Violet was injected to mark the injection site. Only data from rats injected into the RVLM were included for analysis. The data were evaluated using t-tests for paired samples and compared the mean values of vehicle (PBS) against different doses of BNP microinjections. Values of P¦0.05 were considered significant. Microdialysis studies In another set of experiments, a microdialysis probe was lowered unilaterally into the RVLM area using the same coordinates as specified above. After lowering the microdialysis probe into the RVLM, each experiment lasted for 7 h, comprising a 2-h initial stabilization period, 2 h of baseline recordings, 1 h of BNP perfusion and 2 h of post-BNP perfusion. A control group of rats was subjected to similar surgical processes and had the dialysis probe placed in the RVLM and perfused with PBS alone for 7 h. Blood pressure and heart rate measurements were carried out throughout the experiment. Histological verification was performed at the end of each experiment to confirm the exact placement of the microdialysis probe. Two concentric stainless steel tubings were used to construct the microdialysis probe. A 32-gauge inner tube was placed inside a 22-gauge outer tube. A tubular cellulose dialysis membrane (Spectrum) of 300 µm diameter was glued to the outer tube. The length of the dialysis membrane tip was 0.5 mm. The semi-permeable membrane had a mol. wt cut-off of approximately 15,000. The catechols and indole recovery averaged 7% when these probes were tested in vitro at a flow rate of 1.5 µl/min at 25)C. The dialysis probe was perfused with PBS (pH 7.4)32 at the rate of 1.5 µl/min using a microliter syringe pump (Harvard Apparatus, South Natick, MA, U.S.A.) and the initial perfusate of the first 2 h was discarded. Then, a 2-h baseline recording was done before perfusing BNP. Similarly, BNP at a concentration of 40 pmol dissolved in PBS was perfused continuously at a rate of 1.5 µl/min for a period of 1 h, followed by an additional 2 h of perfusion of buffer alone. The perfusion flow was maintained at a constant rate of 1.5 µl/min throughout the experiment. All groups consisted of at least six animals. All the perfusate samples were collected continuously on ice every 15 min. The perfusate was assayed immediately for epinephrine, norepinephrine, dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA). Catechol and indoleamine assays using high-performance liquid chromatography Epinephrine, norepinephrine, DOPAC and 5-HIAA were assayed using a 1 mm#100 mm microbore highperformance liquid chromatography (HPLC) column with electrochemical detector. The HPLC system was comprised of an ISCO 100 DM Syringe Pump and a Series D Pump Controller (Isco, Inc., Lincoln, NB, U.S.A.). The column was 100 mm#1 mm packed with Spherisorb ODS 2 with particle size of 3 µm (Keystone Scientific, Inc., Bellefonte, PA, U.S.A.). An amperometric (LC-4C) dual glassy carbon electrochemical detector (Bioanalytical Systems, Inc., West Lafayette, IN, U.S.A.) set at 0.7 V potential and a Rheodyne injector (Model No. 9125) with an injection loop volume of 5 µl sample were part of the HPLC system

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Fig. 1. Photomicrograph of a coronal section of rat brainstem showing (arrow) the placement of the microdialysis probe in the RVLM. involved in assaying the catechols and indoleamine. The mobile phase consisted of 3.7 g/l citric acid monohydrate, 2.5 g/l anhydrous sodium acetate, 550 mg/l 1-octanesulfonic acid, 350 mg/l disodium EDTA and 5% methanol. The flow rate was 100 µl/min and the pH was 4.3. The dialysis perfusate was assayed immediately for epinephrine, norepinephrine, DOPAC and 5-HIAA every 15 min throughout the baseline period, during the peptide perfusion and for 2 h after the discontinuation of the peptide infusion. Histology At the end of each experiment, the rats were decapitated, the brains removed and stored in 10% formaline for over 10 days. These brains were frozen and 30-µm sections were sliced on a cryostat (5030 Microtome Bright Instruments Company Ltd, Huntingdon, U.K.). These histological sections were stained with Thionin to identify the microdialysis probe placement with reference to the stereotaxic rat brain atlas of Paxinos and Watson.33 Only data obtained from the animals in which the placement of microdialysis probe was in the desired location were included for further analysis. Statistical analyses Perfusate concentrations at each time-point were standardized to the mean baseline concentrations and expressed as a percentage of the baseline value. The blood pressure and heart rate values pertaining to the dialysis experiments were expressed as absolute values. All these values were subjected to statistical analysis using analysis of varianc e for repeated measures followed by a post-hoc Dunnett test.23 RESULTS

Histological identification of the perfusion site The location of the unilateral placement of the microdialysis cannula into the RVLM region is shown in Fig. 1.

Table 1. Mean arterial blood pressure and heart rate in response to unilateral injections (100 nl) of different concentrations of brain natriuretic peptide into the rostral ventrolateral medulla region Blood pressure (mmHg) Baseline PBS BNP 2 pmol 4 pmol 20 pmol 40 pmol

Heart rate (b.p.m.)

77&3 78&3

388&12 380&17

67&2** 64&4** 58&5** 58&3**

328&10** 320&9** 296&12** 262&11**

The baseline blood pressure and heart rate represent the values obtained 2 h after implanting the injection cannula into the RVLM. **P<0.01 compared with PBS (control) injection in each group. Results are mean&S.E.M. (n=6).

Effect of local injection of different doses of brain natriuretic peptide into the rostral ventrolateral medulla Unilateral injections of different doses of BNP into the RVLM and their effects on cardiovascular function are shown in Table 1. Local injections of all the different doses tested resulted in immediate hypotension and bradycardia, which persisted for a couple of minutes before returning to the normal values. Compared to the baseline values (obtained at the end of 2 h after implanting the injection cannula into the RVLM), injection of PBS prior to BNP injections produced no remarkable changes in either blood pressure or heart rate. Though the local injections of 2, 4 and 20 pmol BNP led to a dose-dependent reduction in mean arterial blood pressure, there was no significant difference in blood pressure between

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BP (mmHg) HR (b.p.m.) EN (fmol/5 µl) NE (fmol/5 µl) DOPAC (fmol/5 µl) 5-HIAA (fmol/5 µl)

Hours after initial stabilization period

Initial stabilization period (2 h)

2

3

4

5

81&3 382&15 29&3 38&2 27&4 221&19

78&2 370&12 24&2 37&4 32&6 217&16

76&4 356&17 27&4 37&6 28&7 206&13

80&2 360&14 26&2 38&5 30&3 209&19

78&3 362&9 28&5 35&3 29&6 212&11

For details of treatment, see Experimental Procedures. The blood pressure (BP) and heart rate (HR) values during the initial stabilization period represent the average of eight time intervals of 15 min each. All other values are the average of either eight or four time intervals of 15 min each. EN, epinephrine; NE, norepinephrine. Statistical analysis showed no significant changes. Data are mean&S.E.M. (n=6).

the doses of 20 and 40 pmol. However, the heart rate kept decreasing as the BNP concentration increased. Effect of perfusion of vehicle (phosphate-buffered saline) alone on the cardiovascular and extracellular catechol and indoleamine levels in the rostral ventrolateral medulla As seen in Table 2, the data obtained from the control group of rats which were subjected to similar experimental conditions and had only PBS perfused throughout the experiment showed that neither the cardiovascular functions nor the extracellular catecholamine and indoleamine levels changed significantly. Cardiovascular changes in response to brain natriuretic peptide perfusion into the rostral ventrolateral medulla region Figure 2 shows the changes in blood pressure and heart rate every 15 min before, during and after the perfusion of BNP. Microdialysis perfusion of 40 pmol BNP at the rate of 1.5 µl over a period of 1 h resulted in significant reductions in both blood pressure and heart rate. Effect of brain natriuretic peptide perfusion on the extracellular fluid catechol and indoleamine turnover in the rostral ventrolateral medulla region Changes in catecholamines and indoleamine in response to dialysis perfusion of BNP (40 pmol) are given in Fig. 3. Extracellular concentrations of both epinephrine and norepinephrine decreased from the onset of BNP perfusion and remained depressed for some time, even after termination of the BNP perfusion. In contrast, 5-HIAA levels rose significantly with BNP perfusion and remained significantly elevated for more than 1 h after BNP perfusion. The extracellular DOPAC was unaffected in these experiments.

DISCUSSION

This study defines the neuromodulatory role of BNP (rat BNP-32) in the RVLM and its effect on cardiovascular functions. Direct administration, as well as perfusion of BNP into the RVLM through in vivo microdialysis probes, led to remarkable reductions in both the mean arterial blood pressure and heart rate. Previously, using a similar experimental protocol, the role of ANP on the RVLM was studied and dose-dependent increases in both blood pressure and heart rate were found,6 indicating that ANP and BNP have different effects on the RVLM in regulating cardiovascular functions. However, Ermirio et al.15 have reported functional similarities between ANP and BNP involving microinjections (20 nl of 10"7 M) of these peptides into much more discrete regions of the ventrolateral medulla. The discrepancy could very well be attributed to factors such as the differences in the injection volume and concentrations of these peptides. The BNP used in this study is of much lesser concentration (10"12 M) and larger volume (100 nl). Also, these authors have experimented by injecting ANP into 77 different sites and by injecting BNP into 21 different sites within the ventrolateral medulla, comprising both the RVLM (vasopressor) and the caudal ventrolateral medullary (vasodepressor) regions, whereas we have focused our attention on the whole RVLM region. Furthermore, the differences in the BNP used could also have contributed to the observed differences. We have used rat BNP of 32-amino acid sequence, whereas Ermirio et al.15 used porcine BNP-26. Local injections of different doses of rat BNP-32 into the NTS also produced significant hypotension and bradycardia only in SHRs, whereas the normotensive strains such as the WKY and Sprague– Dawley rats were unaffected.13 It is evident from the present as well as our previous findings13 that rat BNP-32 is region specific, because local application of BNP into the RVLM altered both the blood pressure and heart rate, while injections into the NTS

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Fig. 2. Effect of local perfusion of BNP into the RVLM region and its effect on heart rate (a) and blood pressure (b). BNP at a concentration of 40 pmol dissolved in PBS was perfused at a rate of 1.5 µl/min for a period of 1 h through the in vivo microdialysis probe. Both heart rate and blood pressure showed significant reductions during the BNP perfusion period. Significant values are changes from baseline values. *P<0.05; **P<0.01. Data are mean&S.E.M. (n=6).

failed to elicit any changes in a similar experimental set-up. Intravenous administration of porcine BNP-26 into the deoxycorticosterone acetate hypertensive, SHR and WKY rats resulted in the reduction of only the mean arterial blood pressure, with no marked changes in the heart rate.24,25 The same BNP (porcine BNP-26), when given i.c.v. to the freely moving Wistar rats, altered neither the blood pressure nor the heart rate.41 The circulating form of another BNP, consisting of a 45-amino acid peptide (rat BNP-45), was isolated from rat heart and found to be very distinct from that of porcine BNP in its amino acid sequence.1,21 In rats, the cardiac atria were shown to contain higher levels of BNP-45 than the brain.2 Administration of this peptide intravenously into anesthetized SHRs and WKY rats produced natriuresis and hypotension in these animals, with the SHRs being much more susceptible to BNP-45.26 From these reports, it is not possible to arrive at a clear consensus regarding the role of BNP because of the differences in the BNPs, animal models and the modes of peptide delivery adopted in the above studies. The centrally mediated role of BNP and its interactions with various other vasoactive peptides has been studied. An angiotensin II-induced pressor response was attenuated by the central administration of BNP-26 into freely moving Wistar rats.41 Similarly, secretion of arginine vasopressin was inhibited in response to i.c.v. injection of BNP in the rat.47 Intracerebroventricular injections of BNP

dose-dependently inhibited the central endothelininduced pressor response, plasma catecholamines and adrenocorticotrophic hormone secretion in conscious rats.30 The plasma immunoreactive BNP concentrations showed a profound increase in response to intravenous administration of vasopressin, angiotensin II and endothelin, well-known potent vasopressor agents.18 It is evident from these reports that these peptides not only interact with various neurotransmitter substances, but also interact with one another in maintaining physiological homeostasis. Though the perfusion of BNP into the RVLM significantly altered the extracellular catecholamine and indoleamine concentrations, this study neither addresses the mechanisms by which BNP influences the changes in the neurotransmitters nor offers any explanation pertaining to the actual mechanism(s) involved in the brainstem-mediated hypotensive actions of BNP. Also, it is not clear whether the observed changes in catechols and indole during the BNP perfusion are the result of the direct effect of BNP on the RVLM neurons or secondary to the BNP-induced changes in the peripheral cardiovascular functions. The latter possibility is quite unlikely, because lowering the mean arterial blood pressure by 20 mmHg with intravenous infusion of nitroprusside resulted in increased extracellular catechol and decreased 5-HIAA levels in the RVLM as evaluated independently by both in vivo electrochemical electrode and in vivo microdialysis methods.5,14 Because

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Fig. 3. Altered extracellular epinephrine (a), norepinephrine (b), DOPAC (c) and 5-HIAA (d) of the RVLM region in response to 1-h BNP perfusion into the RVLM region through the in vivo microdialysis probe. The concentrations of both epinephrine and norepinephrine fell during the BNP perfusion and remained significantly lower than the baseline concentration beyond the BNP perfusion period. Basal levels of extracellular epinephrine and norepinephrine were 25&3 and 37&4 fmol/5 µl respectively. DOPAC levels remained more or less stable throughout the experiment. The basal extracellular DOPAC level was 33&6 fmol/5 µl. The concentration of 5-HIAA increased at the onset of BNP perfusion and remained elevated even after the BNP perfusion. The baseline extracellular 5-HIAA level was 203&14 fmol/5µl. Significant values are changes from baseline values. *P<0.05; **P<0.01. Data are mean&S.E.M. (n=6).

norepinephrine release at the nerve terminals can activate presynaptic á2 adrenoceptors to regulate its own release, it would have been expected that the reduced output of norepinephrine following BNP infusion could result in enhanced peripheral transmitter release. However, since the overriding response to BNP infusion is a fall in blood pressure and heart rate, signifying reduced output of norepinephrine peripherally, the results could be

interpreted as indicating a predominance of serotonin-mediated inhibition of norepinephrine. Though the origin of the extracellular epinephrine in the RVLM is not clear, adrenergic neurons from A2 and A5 are reported to project to the RVLM.46 Also, the recurrent collaterals of the C1 neurons7,44 themselves may be the source of the extracellular epinephrine seen in this study. Noradrenergic neurons from the NTS,3,39 area postrema,7 caudal

BNP and rostral ventrolateral medulla

ventrolateral medulla3,16 and A544 are known to have projections to the RVLM. Though the neuronal events leading to the release of epinephrine are unknown, there is a possibility of the noradrenergic neurons having a role in regulating epinephrine release. Serotonergic neuronal projections to the cardiovascular regulatory region of the brainstem have also been well documented.29 The in vivo microdialysis technique is presently the only available technique to sample virtually any substance from almost any brain region. However, the trauma and destruction of brain tissue involved in the process cannot be ignored. Lowering a 300-µmdiameter probe all the way into the RVLM region most certainly damages many of the neurons on its way. Also, the validity of the data obtained from smaller nuclei such as the RVLM with these probes poses another kind of consideration. In this regard, it is noteworthy to add that the probes placed more medially or more laterally resulted in complete disappearance of epinephrine

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and varied levels of norepinephrine. Also, the consistency and reproducibility of the data obtained in different groups of rats strongly suggest that valuable information can still be obtained by carefully and accurately carrying out these experiments.

CONCLUSION

The altered cardiovascular functions in response to the local administration of rat BNP-32 into the RVLM region of urethane-anesthetized Sprague– Dawley rats suggests the possible role of BNP in modulating the metabolic turnover of the monoamines in the RVLM to maintain the vasomotor tone. Acknowledgements—This work was supported by a research grant (MY 025) from Kuwait University to Dr B. R. Dev. We wish to thank Mr Shaji Joseph for his help in plotting the data.

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