Brain Research, 537 (1990) 175-181 Elsevier
175
BIKES 16185
Evidence for clonidine presynaptically modulating amino acid release in the rostral ventral medulla:, role in hypertension Francis D. Tingley III and Stephen P. Arneri6 Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, IL 62702 (U.S.A.) (Accepted 24 July 1990) Key words: Clonidine; a2-Adrenergicreceptor; Spontaneouslyhypertensive rat; Wistar Kyoto rat; y-Aminobutyricacid; Glutamate
Reports suggested that the predominant site of action for the antihypertensive effects of donidine is the rostral ventrolateral medulla (RVL), the presumed tonic vasomotor center. This study examined whether clonidine directly interacts with nerve terminal a2-adrenergic receptors in the RVL to inhibit the release of sympathoexcitatorytransmitters like glutamate (Glu) and aspartate (Asp), and/or facilitate the release of sympathoinhibitorytransmitters like 7-aminobutyricacid (GABA). Release of GABA and Glu was measured from synaptosomes prepared from the rostral ventral medulla of spontaneously hypertensive rats (SHR), a genetic model of hypertension, and normotensive Wistar-Kyoto rats (WKY). Quantification of neurotransmitter release was performed by high-performance liquid chromatography. Depolarization with 35 mM K+ significantly increased by 58-110% the release of GABA, Glu and Asp; however, no strain differences were observed. In contrast, spontaneous release of GABA and Asp was significantlylower in SHR than that of WKY (-36 and -41%, respectively); this effect was not observed for Glu. Clonidine (1 and 10/~M) enhanced the spontaneous release of GABA (+44%), Asp (+50%) and Glu (+70%) in SHR, but not WKY; this effect was prevented by yohimbine (1 ~uM). These data, together with previous findings, support the presence of faeilitory a2-adrenergic receptors on nerve terminals of GABAergic, glutamatergic and aspartaterglc neurons in the rostral ventral medulla. These findings also suggest the existence of another inhibitory transmitter that may mediate the actions of elonidine to decrease sympathetic outflow from the RVL.
INTRODUCTION The predominant site of action for clonidine to lower blood pressure is the medulla oblongata 19'3s'4°. More recent experiments suggest that the active site is localized to the rostral ventrolateral medulla (RVL) 8A°A3"31'39, 42,49. Prevailing dogma suggests that clonidine interacts with a2-adrenergic receptors to reduce the release of norepinephrine (NE) from central catecholaminergic nerve terminals 24,2s,44. However, depletion of catecholamines by pretreatment with reserpine, the tyrosine hydroxylase inhibitor a-methyl-p-tyrosine, and the neurotoxin 6-hydroxydopamine do not appreciably affect the cardiovascular effects of clonidine 12'16'21'22. These data raise the possibility that clonidine may alter the release of non-adrenergic sympathoexcitatory or sympathoinhibitory neurotransmitters in CNS centers for cardiovascular control. The RVL is one of the most critical sites for baroreflex control of arterial pressure (AP) and functions as a major premotor center that generates sympathoexcitatory drive n'29"33-'35'47'5°. Evidence indicates that sympathoexcitatory glutamatergic transmission from rostral struc-
tures, and sympathoinhibitory GABAergic transmission arising from the caudal ventrolateral medulla (CVM), may be crucial to encode and convey baroreceptor information within the R V L 7'15'46"48'53'58'60. In spontaneously hypertensive rats (SHR), a model of experimental hypertension, Arneri6 et al. 3 determined that spontaneous and evoked release of endogenous Glu is notably (2to 4-fold) increased in the RVL of SHR during established hypertension 4. This finding raises the question whether overactive Glu transmission in the RVL increases sympathetic outflow, and whether clonidine may lower AP by affecting Glu and/or G A B A release. Recently, clonidine has been shown to inhibit K ÷evoked release of Glu and G A B A using micropunches of brain containing the RVL 3. However, it was not established whether clonidine interacted directly with presynaptic receptors to regulate the release of Glu or G A B A within the RVL. The present study sought to determine: (1) the synaptic actions of clonidine and the a2-adrenergic receptor antagonist, yohirnbine, on the release of endogenous Glu and G A B A from synaptosomes of the rostral ventral medulla; and (2) whether these interactions were altered in SHR during established hypertension.
Correspondence: S.P. Arnerid. Present address: Abbott Laboratories, AP-10, Neurosciences, Pharmaceutical Discovery, Abbott Park, IL 60064-3500, U.S.A. 0006-8993/90l$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
176
MATERIALS AND METHODS
Spontaneous _~ 12,000
Animals
E
Studies were performed on male Wistar-Kyoto (WKY) and spontaneously hypertensive rats (12-19 weeks old; Harlan, Indianapolis, IN). Animals were given water and standard lab chow ad libitum with a 12 h light/dark schedule (06.00-18.00 h).
C 10,000
K+-Evoked
•k p < 0.05, WKY v s SHR B
WKY
Y
~ P • 0.0,5 S0ont, v s Evoked
~SHR 8,000 E 6,000
Preparation of synaptosomes Rats were killed by decapitation and brains quickly removed and placed in ice-cold 0.32 M sucrose for dissection. The pia-arachnoid membranes were carefully removed, the cerebellum was separated from the hindbrain, and the forebrain excised at the level of the inferior colliculus. A MclIwain tissue chopper was used to coronally cut the remaining hindbrain into slices (0.5 mm thick) starting at calamus scriptorius proceeding in a rostral direction. Slices containing the RVL (+1.0-3.0 mm to calamus) were selected and a horizontal cut was made at the level of the nucleus ambiguus. The ventral aspects of this rostral medulla were then used to prepare synaptosomes. Wet weights were taken on the brain slices and the tissue was homogenized in 20 vols. (v/w) of 0.32 M sucrose using 8 strokes from a Plotter-Elvehjem tissue grinder. Homogenates were centrifuged (10 min, 4 °C, 1500 g) to remove debris and nuclei; supernatant (S1) was carefully decanted and recentrifuged (12 min, 4 °C, 27,000 g). The crude synaptosomal pellet (P2) was then resuspended in 20 vols. (v/original wet wt.) of gassed (95% 02: 5% CO2) Kreb's bicarbonate buffer (in mM): NaC! 118; KCI 5; MgSO 4 1.2; NaHCO 3 25; o-glucose 11; NaH2PO 4 1.2; CaCI 2 1.2; physostigmine 0.1 at pH 7.4, 4 °C. Previous biochemical and ultrastructural evaluations suggest the P2 fractions prepared with this protocol are enriched in synaptosomes2'56.
Release of neurotransminers For release of Asp, Glu and GABA, synaptosomes were exposed to K ÷ (5 or 35 mM Kreb's bicarbonate buffer) with varying concentrations of clonidine (1.0 or 10.0 gM) and 1.0/~M yohimbine for 5 min. The increases in K + concentration were compensated by equiosmolar reductions in Na + concentration. Samples from release were frozen at -20 °C for later analysis of neurotransmitters by high-performance liquid chromatography (HPLC) analysis (Gilson Automated Amino Acid Analyzer) based on the method of Jones et al. 2°, and modified by Arneri6 et al. 5, using precolumn derivatization of amino acids (AA) with o-phthalaldehyde. Separation was achieved by reverse-phase chromatography and fluorescence detection of the A A derivates5'2°. This technique demonstrated a limit of detection of 50 fmol/10 pl of injectate5. Protein content was
4,000
•~ 2,000 _o 0
10
6
GABA
9
6
11
6
11
GLUTAMATE ASPARTATE
6
GABA
10
6
11
6
GLUTAMATE ASPARTATE
Fig. 1. Release of endogenous GABA, Glu and Asp from crude synaptosomes prepared from the rostral ventral medulla of 12-19 week old normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) with established hypertension. Release was measured during spontaneous (5 mM K +) and K+-evoked (35 mM K ÷) conditions. Values are means + S.E.M.; N = replicate experiments; * indicates the value is significantly decreased from WKY, P < 0.05; + indicates the value is significantly increased above the spontaneous conditions, P < 0.05.
determined by using a Coomassie blue dye-binding method 32 with bovine serum albumin as standard.
Statistics Data were represented as the mean + S.E.M. and analyzed by a 2x2×5 split plot analysis of variance (ANOVA) with treatment differences being determined by the Games-Howell test: All others were detected using a paired t-test. The criterion of statistical significance was P < 0.05.
RESULTS
Release of GABA, Glu and Asp Release of endogenous amino acids was measured from normotensive W K Y (SBP = 140 + 3 m m Hg; n = 11) and S H R with established hypertension (SBP = 207 _+ 2 m m Hg; n = 6). W h e n synaptosomes prepared from
TABLE I
Effect of clonidine on the spontaneous release of endogenous GABA, glutamate and aspartate from synaptosomes isolated from the RVL of Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) Values are means +__S.E.M. ;n = 6-11.
Treatments
Endogenous amino acid release (pmol/mg protein~5rain) GA BA
Control (vehicle) Clonidine 10-6 M Clonidine 10-5 M Clonidine 10-5 M + yohimbine 10 6M Yohimbine 10-6 M
Glutamate
Aspartate
WKY
SHR
WKY
SHR
WKY
SHR
4543 + 516 4249 + 727 5434 + 626
2919 + 131" 2986 + 174 4215 + 402**
5480 + 397 5695 + 487 6383 + 765
6333 + 287 7758 + 1245 9525 + 1337"*
3727 + 417 4775 + 470 4684 + 467
2188 + 179" 2964 + 507 3718 + 403**
4719 + 374 5470 + 666
3492 + 696 4299 + 169
6078 + 900 6103 + 669
7466 + 858 7434 + 462
5147 + 687 4221 _+437
3092 + 424 3090 + 308
*P < 0.05, WKY vs SHR; **P < 0.05 treatment vs vehicle.
177 TABLE II Effect of clonidine on the K + evoked release of endogenous GABA, glutamate and aspartate from synaptosomes isolated from the RVL of Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR)
Values are means _+S.E.M.; n = 6-11. Treatments
Evoked amino acid release (pmollmg protein/5 rain) GA BA
Control (vehicle) Clonid ine 10-6 M
Clonidine 10-5 M Clonidine 10-5 M + yohimbine 106 M Yohimbine 10-6M
Glutamate
WKY
SHR
7420 + 944 8438 + 598 6340 + 777
5118 + 119 4734 + 595 4379 + 522
6591 + 838 7414 '_+ 1010
4626 + 453 4193 + 665
WKY
Aspartate SHR
WKY
SHR
9410 + 853 9253 --+1020 8891 + 1105
10760 + 1055 9701 --+1995 9762 _+1699
5880 + 453 5177 _+493 5094 _+572
4594 + 713 4037 + 827 3508 + 626
10530 + 1397 9407 + 1280
11360 + 1110 9356 + 803
4966 + 490 5151 + 462
3676 + 528 3374 + 438
P > 0.05 for WKY vs SHR, and treatment vs vehicle.
the rostral ventral medulla were exposed to 35 mM K + Kreb's buffer there was a significant increase in the evoked release of neurotransmitters (% compared to spontaneous control): G A B A (WKY -- 163%, SHR = 175%), Glu (WKY = 172%, SHR = 170%), and Asp (WKY = 158%, SHR, + 210%). However, no differences were observed between WKY and SHR (P > 0.05). There was a significantly lower (P < 0.05) spontaneous release of G A B A (-36%) and Asp (-41%) in SHR compared to WKY (Fig. 1). These data suggest that in the absence of afferent baroreflex information and local neurotransmitter modulation the spontaneous, but not evoked, release of G A B A and Asp are reduced in SHR. Effects o f clonidine on release When crude synaptosomes were exposed to 1.0 and 10.0/zM clonidine, the drug produced a concentrationdependent increase in spontaneous release of G A B A , Glu, and Asp in SHR, but not WKY (Table I). In the presence of the aE-adrenoceptor antagonist, yohimbine, the effect of clonidine to increase the spontaneous release of Glu, G A B A and Asp was prevented; yohimbine given alone had no effect on release (Table I). In contrast, clonidine caused no significant difference in the evoked release of Glu, G A B A and Asp from control, or between strains (Table II). These data support the suggestion that facilitory aE-adrenergic receptors exist on GABAergic, glutamatergic, and aspartatergic nerve terminals in the rostral ventral medulla.
DISCUSSION This study sought to determine whether clonidine interacted with presynaptic a2-adrenergic receptors on nerve terminals in the rostral ventral medulla to inhibit
release of sympathoexcitatory amino acids like Glu or promote the release of sympathoinhibitory amino acids like G A B A . Instead, the data from this study support the presence of facilitory a2-adrenergic receptors on nerve terminals of GABAergic, glutamatergic and aspartatergic neurons in rostral ventral medulla. Together with previous findings 3, these results implicate the existence of another inhibitory transmitter that may mediate the actions of clonidine to decrease sympathetic outflow. Finally, this study is one of the first reports that disruptions in amino acidergic release processes in the RVL may contribute to the maintenance of hypertension in SHR. Neurotransmission in the R V L Sympathetic outflow is a function of at least 3 major neuronal influences: (1) the intrinsic capacity of RVL neurons to tonically generate vasomotor tone5°; (2) the GABA-mediated baroreceptor inhibition of RVL neurons48; and (3) the influence of rostral structures 17 like
the hypothalamus to send glutamatergic input to modulate sympathetic-related activity generated from the RVL 46. These electrophysiological studies are supported by microinjection studies in the RVL that indicate Glu and other excitatory amino acids increase sympathetic drive, whereas G A B A decreases sympathetic outflow7' 53.57-59 by direct interactions with RVL spinal neurons 28. Moreover, under certain circumstances, other modulatory influences may be expressed, such as a sympathoexcitatory pathway from the nucleus tractus solitarii to the RVL 52. Each of these influences are integrated at the level of RVL neurons. RVL neurons are immunoreactive to antibodies directed against PNMT, Glu, substance P and/or neuropeptide Y, and project to the intermediolateral cell column of the thoracic spinal cord to alter sympathetic preganglionic nerve activity 18'29'3°'33-35.
178 Amino acid neurotransmission in the R V L during hypertension Reports of changes in central amino acid neurotransmission in experimental models of hypertension are few and fragmented 3°. This study indicates that the spontaneous, but not K+-evoked, release of GABA and Asp was reduced in the ventral medulla of SHR. In addition, both spontaneous and evoked release of Glu is unaffected. Consistent with these in vitro data are the following in vivo studies. First, in the RVL of SHR the depressor response to microinjection of GABA is attenuated z3. Second, microinjections of Glu produced equivalent dose-dependent increases in AP in 12-15week-old SHR and WKY 43. Third, bilateral microinjections of tetrodotoxin into the CVM cause a significantly larger increase in WKY, suggesting that tonic GABAmediated sympathoinhibition arising from the CVM is attenuated in SHR 43. However, the depressor response to Glu microinjections into the CVM was greater in SHR 43. This suggests that withdrawal, but not impairment, of GABAergic input to the RVL contributes to the elevation of AP in SHR. Finally, since the intrinsic pacemaker activity of the RVL spinal neurons is not different between WKY and SHR 47, it is plausible that alterations in the release of afferent excitatory or inhibitory amino acid input could result in the initiation, and/or maintenance, of hypertension. Currently, the relationship between noradre:aergic and aminoacidergic neurotransmission during hypertension is poorly understood. Although derangements in noradrenergic neurotransmissions clearly exist with critical central cardiovascular processing centers, such as the NTS and CVM 30'45, recent studies suggest that noradrenergic turnover and release in the RVL is unrelated to arterial pressure changes in SHR 45. These data are consistent with the notion that the medullary cardiovascular actions of clonidine occur independent of direct interactions with ventral medullary catecholaminergic systems. Clonidine's medullary site of action As elaborated in the introduction, the major site of action for clonidine to lower AP is the RVL 8'10"13'39'42'48. This corresponds well with the relative distibution of az-adrenergic receptors localized in human and rat medulla 51. Although clonidine does bind with high affinity to other medullary sites such as the NTS, and can inhibit the release of norepinephrine in the NTS 55, clonidine has little or no effect when microinjected into the NTS a°. Importantly, the area of the ventral medulla used to prepare the synaptosomes used in this study excluded the NTS; however, it did contain midline structures such as the raphe. Despite this loss of anatomical resolution and
Sympathoexcitatory (NTSorHigherCenters)
Sympathoinhibilory (CaudalVentrolateralMedulla)
• /
putative Inhibitor...y
©
Glutamate ~
GABA
)
/ /
__11.
(-)J
~'~
Sympathetic Outflow
Fig. 2. A schematic of the proposed a2-adrenergic receptor sites that clonidine interacts with on non-adrenergic neurons in the rostral ventral medulla to affect sympathoexcitatory and sympathoinhibitory neurotransmission. selectivity (imposed by the difficulty of preparing consistent synaptosomes from RVL punches), it is unlikely that significant contributions were made from these regions since: (1) a2-adrenergic receptors are in very low density in this area of medulla51; and (2) clonidine is ineffective when microinjected into these adjacent regions 1°. Therefore, even though the release effects observed in this study may be quantitatively diluted by the use of adjacent tissue, qualitatively they are likely to represent the actions occuring within RVL. Effects of clonidine on amino acid release Previous work demonstrated that clonidine inhibits the K÷-evoked release of endogenous GABA, Glu and [3H]ACh from micropunches of the RVL via a2-adrenergic receptors 3. Since both Glu and ACh are potent sympathoexcitatory transmitters in the RVL 1'14, these data supported the hypothesis that clonidine lowers AP by decreasing the release of non-adrenergic neurotransmitters that maintain resting AP. However, in contrast to the present study, previous work was performed only in normotensive WKY rats and the effects of clonidine were on K÷-evoked, not spontaneous, release. It should be kept in mind that relative to the synaptosomal preparation, micropunches still preserve a significant amount of synaptic organization and would permit the expression of multisynaptic interactions before eliciting a net response. Results of the two studies suggest that the neuronal
179 relationships in the R V L m a y be m o r e complex than previously thought. A schematic of the p r o p o s e d ct2adrenergic r e c e p t o r sites that clonidine interacts with on n o n - a d r e n e r g i c neurons in the rostral ventral m e d u l l a is shown in Fig. 2. Confirmation of these interactions would reconcile most of the differences b e t w e e n the two studies. T h e ability of clonidine to m o d u l a t e sympathetic outflow from the R V L is d e p e n d e n t on its actions both on s y m p a t h o e x c i t a t o r y and sympathoinhibitory influences (Fig. 2). H o w e v e r , the actions of clonidine to affect the release of individual neurotransmitters is d e p e n d e n t on the juxtaposition of afferent neuronal influences and the c o r r e s p o n d i n g presence of a2-adrenergic receptors. In s y n a p t o s o m a l p r e p a r a t i o n s the spontaneous release of G A B A , A s p and Glu is a p p r o x i m a t e l y 10-fold greater than intact micropunches. These data support the idea that a putative inhibitory substance tonically suppresses the release of these transmitters, but is unable to do so when the neuronal circuits are disrupted. In micropunches activation of a2-adrenergic receptors both enhance the release of amino acids as well as the putative inhibitory transmitter. H o w e v e r , the governing influence of this yet to be identified transmitter p r e d o m i n a t e s
during neuronal d e p o l a r i z a t i o n and yields the p a t t e r n of release o b s e r v e d in the intact slice. T h e s e slice d a t a are consistent with the p a t t e r n of neurotransmission that would be p r e d i c t e d to lower A P without altering baroreflex gain 54. F u r t h e r e x p e r i m e n t s are in progress to establish w h e t h e r this p a t t e r n is also o b s e r v e d in vivo. Currently the identity of the putative inhibitory transmitter is unknown. H o w e v e r , it is interesting to speculate that it m a y be r e l a t e d to the recently described substance isolated from calf brain, a 'clonidine displacing substance' (CDS) that acts at ' i m i d a z o l i n e - p e r f e r r i n g ' sites in the R V L 6"27. A l r e a d y contrasting results exist concerning the actions of C D S microinjected into the ventral m e d u l l a 9' 26. Resolving these a p p a r e n t conflicts and establishing a n e u r o t r a n s m i t t e r function for C D S should provide new insight into the central antihypertensive actions of clonidine and related drugs.
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39
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55
56
of 2-(2,6-dichlorphenylamino)-2-imidazoline hydrochloride (St 155) after infusion into the cat's vertebral artery, Eur. J. Pharmacol., 2 (1967) 9-13. Schlafke, M. and Loeschke, H.H., Lokalisation eines an der Regulation von Atmung und Kreislauf beteiligten Gebietes an der ventralen Oberflache der Medulla oblongata durch Kalteblockade, Pflfigers Arch. Ges. Physiol., 297 (1967) 201-220. Schmitt, H. and Schmitt H., Localization of the hypotensive effect of 2-(2,6-dichlorphenylamino)-2-imidazoline hydrochloride (St. 155, Catapresan), Eur. J. Pharmacol., 6 (1969) 8-12. Sinha, J.N., Tangri, K.K., Bhargava, K.P. and Schmitt, H., Central sites of sympathoinhibitory effects of clonidine and L-dopa. In P. Milliez and M. Safar, (Eds.), Recent Advances in Hypertension, Boehringer, lngelheim, 1975, pp. 97-109. Sinha, J.N., Gurtu, S. and Sharma, D.K., An analysis of the a-adrenoceptor modulation of vasomotor tone at the level of lateral medullary pressor area (LMPA), Naunyn-Schmiedeberg's Arch. Pharmacol., 330 (1985) 163-168. Smith, J.K. and Barron, K.W., The rostral and caudal ventrolateral medulla in young spontaneously hypertensive rats, Brain Research, 506 (1990) 153-158. Starke, K. and Montel, H., Involvement of alpha-receptors in clonidine-induced inhibition of transmitter release from central monoamine neurons, Int. J. Neuropharmacol., 12 (1973) 10731080. Stephenson, L.L., Cline, W.H. and Arneric, S.P., Noradrenergic transmission in the nucleus tractus solitarius (NTS), rostral ventrolateral medulla (RVL) and caudal ventrolateral medulla (CVM) of normotensive Wistar Kyoto rats (WKY) and spontaneously hypertensive (SHR), FASEB J. 3 (1989) A408. Sun, M.-K. and Guyenet, P.G., Hypothalamic glutamatergic input to medullary sympathoexcitatory neurons in rats, Am. J. Physiol., 251 (1986) R798-R810. Sun, M.-K. and Guyenet, P.G., Medullospinal sympathoexcitatory neurons in normotensive and spontaneously hypertensive rats, Am. J. Physiol., 250 (1986) R910-R917. Sun, M.K. and Guyenet, P.G., GABA-mediated baroreceptor inhibition of reticulospinal neurons in rats, Am. J. Physiol., 249 (1986) R672-R680. Sun, M.K. and Guyenet, P.G., Effect of clonidine and yaminobutyric acid on the discharges of medullo-spinal sympathoexcitatory neurons in the rat, Brain Research, 368 (1986) 1-17. Sun, M.-K., Young, B.S., Hackett, J.T. and Guyenet, P:G., Rostral ventrolateral medullary neurons with intrinsic pacemaker properties are not catecholaminergic, Brain Research, 451 (1988) 345-349. Unnerstall, J.R., Kopajtic, T.A. and Kuhar, M.J., Distribution of alpha-2 agonist binding sites in the rat and human central nervous system: analysis of some functional, anatomical correlates of the pharmacological effects of clonidine and related adrenergic agents, Brain Res. Rev., 7 (1984) 79-101. Urbanski, R. and Sapru, H.N., Evidence for a sympathoexcitatory pathway from the nucleus tractus solitarii to the ventrolateral medullary pressor area, J. Auton. Nerv. Syst., 23 (1988) 161-174. Urbanski, R.W. and Sapru, H.N., Putative neurotransmitters involved in medullary cardiovascular regulation, J. Auton. Nerv. Syst., 25 (1988) 181-193. Watkins, L. and Maixner, W., Clonidine's antihypertensive effect is not associated with enhanced baroreflex-mediated bradycardia, Soc. Neurosci. Abstr., 15 (1989) 467.12. Wemer, J., Frankhuyzen, A.L. and Muider, A.H., Pharmacological characterization of presynaptic alpha-adrenoceptors in the nucleus tractus solitarii and the cerebral cortex of the rat, Neuropharmacology, 21 (1982) 499-506. Whittaker, V.P. and Barker, L.A. The subcellular fractionation of brain tissue with special reference to the preparation of synaptosomes and their component organelles, Methods Neurochem., 2 (1972) 1-52.
181 57 Willette, R.N., Barcas, P.P., Krieger, A.J. and Sapru, H.N., Vasopressor depressor areas in the rat medulla. Identification by microinjection of L-glutamate, Neuropharmacology, 22 (1983) 1071-1079. 58 Willette, R.N., Bareas, P.E, Krieger, A.J. and Sapru, H.N., Endogenous GABAergic mechanisms in the medulla and the regulation of blood pressure, J. Pharmacol. Exp. Ther., 230 (1984) 34-39.
59 Willette, R.N., Krieger, A.J., Barcas, P.P. and Sapru, H.N., Medullary gamma-aminobutyric acid (GABA) receptors and the regulation of blood pressure in the rat, J. Pharmacol. Exp. Ther., 226 (1983) 893-899. 60 Willette, R.N., Punnen, S., Krieger, A.J. and Sapru, H.N., Interdependence of rostral and caudal ventrolateral medullary areas in the control of blood pressure, Brain Research, 321 (1984) 169-174.
175
BIKES 16185
Evidence for clonidine presynaptically modulating amino acid release in the rostral ventral medulla:, role in hypertension Francis D. Tingley III and Stephen P. Arneri6 Department of Pharmacology, Southern Illinois University School of Medicine, Springfield, IL 62702 (U.S.A.) (Accepted 24 July 1990) Key words: Clonidine; a2-Adrenergicreceptor; Spontaneouslyhypertensive rat; Wistar Kyoto rat; y-Aminobutyricacid; Glutamate
Reports suggested that the predominant site of action for the antihypertensive effects of donidine is the rostral ventrolateral medulla (RVL), the presumed tonic vasomotor center. This study examined whether clonidine directly interacts with nerve terminal a2-adrenergic receptors in the RVL to inhibit the release of sympathoexcitatorytransmitters like glutamate (Glu) and aspartate (Asp), and/or facilitate the release of sympathoinhibitorytransmitters like 7-aminobutyricacid (GABA). Release of GABA and Glu was measured from synaptosomes prepared from the rostral ventral medulla of spontaneously hypertensive rats (SHR), a genetic model of hypertension, and normotensive Wistar-Kyoto rats (WKY). Quantification of neurotransmitter release was performed by high-performance liquid chromatography. Depolarization with 35 mM K+ significantly increased by 58-110% the release of GABA, Glu and Asp; however, no strain differences were observed. In contrast, spontaneous release of GABA and Asp was significantlylower in SHR than that of WKY (-36 and -41%, respectively); this effect was not observed for Glu. Clonidine (1 and 10/~M) enhanced the spontaneous release of GABA (+44%), Asp (+50%) and Glu (+70%) in SHR, but not WKY; this effect was prevented by yohimbine (1 ~uM). These data, together with previous findings, support the presence of faeilitory a2-adrenergic receptors on nerve terminals of GABAergic, glutamatergic and aspartaterglc neurons in the rostral ventral medulla. These findings also suggest the existence of another inhibitory transmitter that may mediate the actions of elonidine to decrease sympathetic outflow from the RVL.
INTRODUCTION The predominant site of action for clonidine to lower blood pressure is the medulla oblongata 19'3s'4°. More recent experiments suggest that the active site is localized to the rostral ventrolateral medulla (RVL) 8A°A3"31'39, 42,49. Prevailing dogma suggests that clonidine interacts with a2-adrenergic receptors to reduce the release of norepinephrine (NE) from central catecholaminergic nerve terminals 24,2s,44. However, depletion of catecholamines by pretreatment with reserpine, the tyrosine hydroxylase inhibitor a-methyl-p-tyrosine, and the neurotoxin 6-hydroxydopamine do not appreciably affect the cardiovascular effects of clonidine 12'16'21'22. These data raise the possibility that clonidine may alter the release of non-adrenergic sympathoexcitatory or sympathoinhibitory neurotransmitters in CNS centers for cardiovascular control. The RVL is one of the most critical sites for baroreflex control of arterial pressure (AP) and functions as a major premotor center that generates sympathoexcitatory drive n'29"33-'35'47'5°. Evidence indicates that sympathoexcitatory glutamatergic transmission from rostral struc-
tures, and sympathoinhibitory GABAergic transmission arising from the caudal ventrolateral medulla (CVM), may be crucial to encode and convey baroreceptor information within the R V L 7'15'46"48'53'58'60. In spontaneously hypertensive rats (SHR), a model of experimental hypertension, Arneri6 et al. 3 determined that spontaneous and evoked release of endogenous Glu is notably (2to 4-fold) increased in the RVL of SHR during established hypertension 4. This finding raises the question whether overactive Glu transmission in the RVL increases sympathetic outflow, and whether clonidine may lower AP by affecting Glu and/or G A B A release. Recently, clonidine has been shown to inhibit K ÷evoked release of Glu and G A B A using micropunches of brain containing the RVL 3. However, it was not established whether clonidine interacted directly with presynaptic receptors to regulate the release of Glu or G A B A within the RVL. The present study sought to determine: (1) the synaptic actions of clonidine and the a2-adrenergic receptor antagonist, yohirnbine, on the release of endogenous Glu and G A B A from synaptosomes of the rostral ventral medulla; and (2) whether these interactions were altered in SHR during established hypertension.
Correspondence: S.P. Arnerid. Present address: Abbott Laboratories, AP-10, Neurosciences, Pharmaceutical Discovery, Abbott Park, IL 60064-3500, U.S.A. 0006-8993/90l$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
176
MATERIALS AND METHODS
Spontaneous _~ 12,000
Animals
E
Studies were performed on male Wistar-Kyoto (WKY) and spontaneously hypertensive rats (12-19 weeks old; Harlan, Indianapolis, IN). Animals were given water and standard lab chow ad libitum with a 12 h light/dark schedule (06.00-18.00 h).
C 10,000
K+-Evoked
•k p < 0.05, WKY v s SHR B
WKY
Y
~ P • 0.0,5 S0ont, v s Evoked
~SHR 8,000 E 6,000
Preparation of synaptosomes Rats were killed by decapitation and brains quickly removed and placed in ice-cold 0.32 M sucrose for dissection. The pia-arachnoid membranes were carefully removed, the cerebellum was separated from the hindbrain, and the forebrain excised at the level of the inferior colliculus. A MclIwain tissue chopper was used to coronally cut the remaining hindbrain into slices (0.5 mm thick) starting at calamus scriptorius proceeding in a rostral direction. Slices containing the RVL (+1.0-3.0 mm to calamus) were selected and a horizontal cut was made at the level of the nucleus ambiguus. The ventral aspects of this rostral medulla were then used to prepare synaptosomes. Wet weights were taken on the brain slices and the tissue was homogenized in 20 vols. (v/w) of 0.32 M sucrose using 8 strokes from a Plotter-Elvehjem tissue grinder. Homogenates were centrifuged (10 min, 4 °C, 1500 g) to remove debris and nuclei; supernatant (S1) was carefully decanted and recentrifuged (12 min, 4 °C, 27,000 g). The crude synaptosomal pellet (P2) was then resuspended in 20 vols. (v/original wet wt.) of gassed (95% 02: 5% CO2) Kreb's bicarbonate buffer (in mM): NaC! 118; KCI 5; MgSO 4 1.2; NaHCO 3 25; o-glucose 11; NaH2PO 4 1.2; CaCI 2 1.2; physostigmine 0.1 at pH 7.4, 4 °C. Previous biochemical and ultrastructural evaluations suggest the P2 fractions prepared with this protocol are enriched in synaptosomes2'56.
Release of neurotransminers For release of Asp, Glu and GABA, synaptosomes were exposed to K ÷ (5 or 35 mM Kreb's bicarbonate buffer) with varying concentrations of clonidine (1.0 or 10.0 gM) and 1.0/~M yohimbine for 5 min. The increases in K + concentration were compensated by equiosmolar reductions in Na + concentration. Samples from release were frozen at -20 °C for later analysis of neurotransmitters by high-performance liquid chromatography (HPLC) analysis (Gilson Automated Amino Acid Analyzer) based on the method of Jones et al. 2°, and modified by Arneri6 et al. 5, using precolumn derivatization of amino acids (AA) with o-phthalaldehyde. Separation was achieved by reverse-phase chromatography and fluorescence detection of the A A derivates5'2°. This technique demonstrated a limit of detection of 50 fmol/10 pl of injectate5. Protein content was
4,000
•~ 2,000 _o 0
10
6
GABA
9
6
11
6
11
GLUTAMATE ASPARTATE
6
GABA
10
6
11
6
GLUTAMATE ASPARTATE
Fig. 1. Release of endogenous GABA, Glu and Asp from crude synaptosomes prepared from the rostral ventral medulla of 12-19 week old normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) with established hypertension. Release was measured during spontaneous (5 mM K +) and K+-evoked (35 mM K ÷) conditions. Values are means + S.E.M.; N = replicate experiments; * indicates the value is significantly decreased from WKY, P < 0.05; + indicates the value is significantly increased above the spontaneous conditions, P < 0.05.
determined by using a Coomassie blue dye-binding method 32 with bovine serum albumin as standard.
Statistics Data were represented as the mean + S.E.M. and analyzed by a 2x2×5 split plot analysis of variance (ANOVA) with treatment differences being determined by the Games-Howell test: All others were detected using a paired t-test. The criterion of statistical significance was P < 0.05.
RESULTS
Release of GABA, Glu and Asp Release of endogenous amino acids was measured from normotensive W K Y (SBP = 140 + 3 m m Hg; n = 11) and S H R with established hypertension (SBP = 207 _+ 2 m m Hg; n = 6). W h e n synaptosomes prepared from
TABLE I
Effect of clonidine on the spontaneous release of endogenous GABA, glutamate and aspartate from synaptosomes isolated from the RVL of Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) Values are means +__S.E.M. ;n = 6-11.
Treatments
Endogenous amino acid release (pmol/mg protein~5rain) GA BA
Control (vehicle) Clonidine 10-6 M Clonidine 10-5 M Clonidine 10-5 M + yohimbine 10 6M Yohimbine 10-6 M
Glutamate
Aspartate
WKY
SHR
WKY
SHR
WKY
SHR
4543 + 516 4249 + 727 5434 + 626
2919 + 131" 2986 + 174 4215 + 402**
5480 + 397 5695 + 487 6383 + 765
6333 + 287 7758 + 1245 9525 + 1337"*
3727 + 417 4775 + 470 4684 + 467
2188 + 179" 2964 + 507 3718 + 403**
4719 + 374 5470 + 666
3492 + 696 4299 + 169
6078 + 900 6103 + 669
7466 + 858 7434 + 462
5147 + 687 4221 _+437
3092 + 424 3090 + 308
*P < 0.05, WKY vs SHR; **P < 0.05 treatment vs vehicle.
177 TABLE II Effect of clonidine on the K + evoked release of endogenous GABA, glutamate and aspartate from synaptosomes isolated from the RVL of Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR)
Values are means _+S.E.M.; n = 6-11. Treatments
Evoked amino acid release (pmollmg protein/5 rain) GA BA
Control (vehicle) Clonid ine 10-6 M
Clonidine 10-5 M Clonidine 10-5 M + yohimbine 106 M Yohimbine 10-6M
Glutamate
WKY
SHR
7420 + 944 8438 + 598 6340 + 777
5118 + 119 4734 + 595 4379 + 522
6591 + 838 7414 '_+ 1010
4626 + 453 4193 + 665
WKY
Aspartate SHR
WKY
SHR
9410 + 853 9253 --+1020 8891 + 1105
10760 + 1055 9701 --+1995 9762 _+1699
5880 + 453 5177 _+493 5094 _+572
4594 + 713 4037 + 827 3508 + 626
10530 + 1397 9407 + 1280
11360 + 1110 9356 + 803
4966 + 490 5151 + 462
3676 + 528 3374 + 438
P > 0.05 for WKY vs SHR, and treatment vs vehicle.
the rostral ventral medulla were exposed to 35 mM K + Kreb's buffer there was a significant increase in the evoked release of neurotransmitters (% compared to spontaneous control): G A B A (WKY -- 163%, SHR = 175%), Glu (WKY = 172%, SHR = 170%), and Asp (WKY = 158%, SHR, + 210%). However, no differences were observed between WKY and SHR (P > 0.05). There was a significantly lower (P < 0.05) spontaneous release of G A B A (-36%) and Asp (-41%) in SHR compared to WKY (Fig. 1). These data suggest that in the absence of afferent baroreflex information and local neurotransmitter modulation the spontaneous, but not evoked, release of G A B A and Asp are reduced in SHR. Effects o f clonidine on release When crude synaptosomes were exposed to 1.0 and 10.0/zM clonidine, the drug produced a concentrationdependent increase in spontaneous release of G A B A , Glu, and Asp in SHR, but not WKY (Table I). In the presence of the aE-adrenoceptor antagonist, yohimbine, the effect of clonidine to increase the spontaneous release of Glu, G A B A and Asp was prevented; yohimbine given alone had no effect on release (Table I). In contrast, clonidine caused no significant difference in the evoked release of Glu, G A B A and Asp from control, or between strains (Table II). These data support the suggestion that facilitory aE-adrenergic receptors exist on GABAergic, glutamatergic, and aspartatergic nerve terminals in the rostral ventral medulla.
DISCUSSION This study sought to determine whether clonidine interacted with presynaptic a2-adrenergic receptors on nerve terminals in the rostral ventral medulla to inhibit
release of sympathoexcitatory amino acids like Glu or promote the release of sympathoinhibitory amino acids like G A B A . Instead, the data from this study support the presence of facilitory a2-adrenergic receptors on nerve terminals of GABAergic, glutamatergic and aspartatergic neurons in rostral ventral medulla. Together with previous findings 3, these results implicate the existence of another inhibitory transmitter that may mediate the actions of clonidine to decrease sympathetic outflow. Finally, this study is one of the first reports that disruptions in amino acidergic release processes in the RVL may contribute to the maintenance of hypertension in SHR. Neurotransmission in the R V L Sympathetic outflow is a function of at least 3 major neuronal influences: (1) the intrinsic capacity of RVL neurons to tonically generate vasomotor tone5°; (2) the GABA-mediated baroreceptor inhibition of RVL neurons48; and (3) the influence of rostral structures 17 like
the hypothalamus to send glutamatergic input to modulate sympathetic-related activity generated from the RVL 46. These electrophysiological studies are supported by microinjection studies in the RVL that indicate Glu and other excitatory amino acids increase sympathetic drive, whereas G A B A decreases sympathetic outflow7' 53.57-59 by direct interactions with RVL spinal neurons 28. Moreover, under certain circumstances, other modulatory influences may be expressed, such as a sympathoexcitatory pathway from the nucleus tractus solitarii to the RVL 52. Each of these influences are integrated at the level of RVL neurons. RVL neurons are immunoreactive to antibodies directed against PNMT, Glu, substance P and/or neuropeptide Y, and project to the intermediolateral cell column of the thoracic spinal cord to alter sympathetic preganglionic nerve activity 18'29'3°'33-35.
178 Amino acid neurotransmission in the R V L during hypertension Reports of changes in central amino acid neurotransmission in experimental models of hypertension are few and fragmented 3°. This study indicates that the spontaneous, but not K+-evoked, release of GABA and Asp was reduced in the ventral medulla of SHR. In addition, both spontaneous and evoked release of Glu is unaffected. Consistent with these in vitro data are the following in vivo studies. First, in the RVL of SHR the depressor response to microinjection of GABA is attenuated z3. Second, microinjections of Glu produced equivalent dose-dependent increases in AP in 12-15week-old SHR and WKY 43. Third, bilateral microinjections of tetrodotoxin into the CVM cause a significantly larger increase in WKY, suggesting that tonic GABAmediated sympathoinhibition arising from the CVM is attenuated in SHR 43. However, the depressor response to Glu microinjections into the CVM was greater in SHR 43. This suggests that withdrawal, but not impairment, of GABAergic input to the RVL contributes to the elevation of AP in SHR. Finally, since the intrinsic pacemaker activity of the RVL spinal neurons is not different between WKY and SHR 47, it is plausible that alterations in the release of afferent excitatory or inhibitory amino acid input could result in the initiation, and/or maintenance, of hypertension. Currently, the relationship between noradre:aergic and aminoacidergic neurotransmission during hypertension is poorly understood. Although derangements in noradrenergic neurotransmissions clearly exist with critical central cardiovascular processing centers, such as the NTS and CVM 30'45, recent studies suggest that noradrenergic turnover and release in the RVL is unrelated to arterial pressure changes in SHR 45. These data are consistent with the notion that the medullary cardiovascular actions of clonidine occur independent of direct interactions with ventral medullary catecholaminergic systems. Clonidine's medullary site of action As elaborated in the introduction, the major site of action for clonidine to lower AP is the RVL 8'10"13'39'42'48. This corresponds well with the relative distibution of az-adrenergic receptors localized in human and rat medulla 51. Although clonidine does bind with high affinity to other medullary sites such as the NTS, and can inhibit the release of norepinephrine in the NTS 55, clonidine has little or no effect when microinjected into the NTS a°. Importantly, the area of the ventral medulla used to prepare the synaptosomes used in this study excluded the NTS; however, it did contain midline structures such as the raphe. Despite this loss of anatomical resolution and
Sympathoexcitatory (NTSorHigherCenters)
Sympathoinhibilory (CaudalVentrolateralMedulla)
• /
putative Inhibitor...y
©
Glutamate ~
GABA
)
/ /
__11.
(-)J
~'~
Sympathetic Outflow
Fig. 2. A schematic of the proposed a2-adrenergic receptor sites that clonidine interacts with on non-adrenergic neurons in the rostral ventral medulla to affect sympathoexcitatory and sympathoinhibitory neurotransmission. selectivity (imposed by the difficulty of preparing consistent synaptosomes from RVL punches), it is unlikely that significant contributions were made from these regions since: (1) a2-adrenergic receptors are in very low density in this area of medulla51; and (2) clonidine is ineffective when microinjected into these adjacent regions 1°. Therefore, even though the release effects observed in this study may be quantitatively diluted by the use of adjacent tissue, qualitatively they are likely to represent the actions occuring within RVL. Effects of clonidine on amino acid release Previous work demonstrated that clonidine inhibits the K÷-evoked release of endogenous GABA, Glu and [3H]ACh from micropunches of the RVL via a2-adrenergic receptors 3. Since both Glu and ACh are potent sympathoexcitatory transmitters in the RVL 1'14, these data supported the hypothesis that clonidine lowers AP by decreasing the release of non-adrenergic neurotransmitters that maintain resting AP. However, in contrast to the present study, previous work was performed only in normotensive WKY rats and the effects of clonidine were on K÷-evoked, not spontaneous, release. It should be kept in mind that relative to the synaptosomal preparation, micropunches still preserve a significant amount of synaptic organization and would permit the expression of multisynaptic interactions before eliciting a net response. Results of the two studies suggest that the neuronal
179 relationships in the R V L m a y be m o r e complex than previously thought. A schematic of the p r o p o s e d ct2adrenergic r e c e p t o r sites that clonidine interacts with on n o n - a d r e n e r g i c neurons in the rostral ventral m e d u l l a is shown in Fig. 2. Confirmation of these interactions would reconcile most of the differences b e t w e e n the two studies. T h e ability of clonidine to m o d u l a t e sympathetic outflow from the R V L is d e p e n d e n t on its actions both on s y m p a t h o e x c i t a t o r y and sympathoinhibitory influences (Fig. 2). H o w e v e r , the actions of clonidine to affect the release of individual neurotransmitters is d e p e n d e n t on the juxtaposition of afferent neuronal influences and the c o r r e s p o n d i n g presence of a2-adrenergic receptors. In s y n a p t o s o m a l p r e p a r a t i o n s the spontaneous release of G A B A , A s p and Glu is a p p r o x i m a t e l y 10-fold greater than intact micropunches. These data support the idea that a putative inhibitory substance tonically suppresses the release of these transmitters, but is unable to do so when the neuronal circuits are disrupted. In micropunches activation of a2-adrenergic receptors both enhance the release of amino acids as well as the putative inhibitory transmitter. H o w e v e r , the governing influence of this yet to be identified transmitter p r e d o m i n a t e s
during neuronal d e p o l a r i z a t i o n and yields the p a t t e r n of release o b s e r v e d in the intact slice. T h e s e slice d a t a are consistent with the p a t t e r n of neurotransmission that would be p r e d i c t e d to lower A P without altering baroreflex gain 54. F u r t h e r e x p e r i m e n t s are in progress to establish w h e t h e r this p a t t e r n is also o b s e r v e d in vivo. Currently the identity of the putative inhibitory transmitter is unknown. H o w e v e r , it is interesting to speculate that it m a y be r e l a t e d to the recently described substance isolated from calf brain, a 'clonidine displacing substance' (CDS) that acts at ' i m i d a z o l i n e - p e r f e r r i n g ' sites in the R V L 6"27. A l r e a d y contrasting results exist concerning the actions of C D S microinjected into the ventral m e d u l l a 9' 26. Resolving these a p p a r e n t conflicts and establishing a n e u r o t r a n s m i t t e r function for C D S should provide new insight into the central antihypertensive actions of clonidine and related drugs.
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