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Endogenous GABA acts on GABAB receptors in nucleus tractus solitarius to increase blood pressure
Brain Research, 526 (1990) 235-240 Elsevier
235
BRES 15809
Endogenous G A B A acts on GABAB receptors in nucleus tractus solitarius to increase blood pressure Alan E Sved and Judith C. Sved Department of Behavioral Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260 (U.S.A.) (Accepted 6 March 1990) Key words: Phaclofen; Nipecotic acid; 7-Aminobutyric acid transaminase inhibitor; Baclofen; Hypertension
Previous studies found that injection of the G A B A uptake inhibitor nipecotic acid into the nucleus tractus solitarius (NTS) increases arterial pressure. This effect of nipecotic acid was not antagonized by the selective G A B A A receptor blocking agent bicuculline, suggesting that the action of nipecotic acid was mediated through an action of G A B A on G A B A B receptors in the NTS. The present studies examined this issue using a newly described G A B A s antagonist, phaclofen. Injection of phaclofen (4 nmol in 100 nl artificial CSF) into the NTS of chloralose-anesthetized rats produced a slight decrease in arterial pressure (-8 + 2 mmHg) lasting less than 1 min. Smaller doses had no effect. Phaclofen antagonized in a dose-dependent (0.5-4 nmol) manner the increase in arterial pressure produced by injection into the NTS of the G A B A n agonist baclofen (5-100 pmol). In contrast, phaclofen had no effect on the pressor response elicited by injection into the NTS of the G A B A A agonist muscimol. Phaclofen (4 nmol) injected into the NTS totally reversed the increase in blood pressure elicited by injection into the NTS of a maximally effective dose of nipecotic acid (10 nmol). Phaclofen also inhibited the pressor response elicited by injection into the NTS of another indirectly acting G A B A agonist, ?-vinylGABA (GVG). Although GVG is an effective inhibitor of G A B A transaminase, the enzyme involved in the metabolism of GABA, the time course of inhibition of G A B A transaminase evoked by GVG was not consistent with the pressor response being produced by this mechanism. However, the pressor response elicited by GVG is consistent with its reported ability to inhibit G A B A uptake. These results support the hypothesis that G A B A tonically released in the NTS acts specifically on G A B A n receptors to increase blood pressure.
INTRODUCTION The nucleus tractus solitarius (NTS), the site of termination within the brain of baroreceptor afferent fibers 12, has repeatedly been shown to be involved in the regulation of arterial pressure (AP) 22. Many neuroactive substances present within the NTS have been shown to be involved in this process (e.g. refs. 1, 2, 3, 6, 21 and 24), although the manner in which any specific neurotransmitter acts physiologically in the regulation of AP has not been completely elucidated. We have previously demonstrated that injection into the NTS of nipecotic acid (NIP), a selective inhibitor of G A B A uptake 5'15-18, produces an increase in A P 3'23. The most straight-forward interpretation of this finding is that potentiating the action of synaptically released G A B A in the NTS, by blocking its clearance from the synapse by uptake, elicits an increase in AP. Since selective stimulation of either G A B A A or G A B A B receptors in the NTS can elicit a pressor response 3'23, G A B A tonically released into synapses in the NTS may normally act on either or both of these receptors. Recent studies 23 indicate that the pressor response evoked by injection of
NIP into the NTS is not attenuated by the selective G A B A A antagonist bicuculline (BIC), suggesting that G A B A may normally act on G A B A B receptors in the NTS to maintain AP. At the time those studies were conducted, no selective G A B A a antagonists were available. However, a series of baclofen derivatives that act as selective antagonists of the G A B A B receptor have recently been described 13'14, with the phosphonic acid derivative of baclofen (BAC), phaclofen (PHAC) being the prototypic agent. The present studies examined the effect of administering PHAC into the NTS on baseline AP and the increase in AP elicited by stimulation of G A B A receptors in the NTS. The results support our previous suggestion that potentiating the action of tonically released G A B A in the NTS elevates AP through stimulation of G A B A B receptors. MATERIALS AND METHODS Adult male Sprague-Dawley rats, 300-450 g (Zivic-Miller, Allison Park, PA) were housed in single cages in a temperaturecontrolled room on a 12 h light/dark cycle with food and tap water available ad libitum for at least 1 week prior to use in experiments.
Correspondence: A. Sved, Department of Behavioral Neuroscience, University of Pittsburgh, 446 Crawford Hall, Pittsburgh, PA 15260, U.S.A. D~fi-ROO~/O~/lflq ~if)(~ IOCKIl=;l~vi~r ~eienr~e Pnhli~her~ B V (Riomedica! r)ivision)
236 Rats were anesthetized with halothane (2% in 100% 02 administered through a nose cone). A cannula (PE 50 tubing filled with heparinized saline) was inserted in the right femoral artery for recording arterial pressure (AP), mean arterial pressure (MAP) and heart rate (HR) (Grass Model 7 Physiograph). A second cannula was placed in the right femoral vein for administering drugs. The trachea was cannulated and rats artificially ventilated with 2% halothane in 100% 0 2 (Harvard Small Animal Respirator) following the administration of a muscle relaxant (d-tubocurarine, 0.5 mg/kg, i.v., supplemented hourly with 0.2 mg/kg, i.v.). Rats were placed in a stereotaxic instrument (Kopf) with the incisor bar positioned 11 mm below the interaural line. The dorsal surface of the brain stem was exposed by limited craniotomy and, with the aid of a surgical microscope, the area postrema visualized. a-Chloralose was administered (60 mg/kg, i.v., supplemented hourly with 20 mg/kg, i.v.) and the halothane terminated. Rats were ventilated with 100% O 2 throughout the remainder of the experiment. Blood gases were measured (IL model 1304 blood gas analyzer) periodically and the respirator was adjusted to maintain arterial pCO 2 in the range of 38-45 mm Hg. Injections of GABA agents were made into the NTS using single or double barrel glass micropipettes pulled to an outer diameter of 40-50/~m. Double barrel pipettes were used in all experiments in which an agonist and antagonist were injected into the same site. The tip of the micropipette was positioned 0.5 mm rostral to the calamus scriptorius, 0.5 mm lateral from the midline and 0.5 mm deep to the surface of the brain stem for injection into the NTS. All drugs were dissolved in artificial cerebrospinal fluid (aCSF (in mM): NaCi 128, KCI 2.6, CaC! 2 1.3, MgCl 2 0.9, NaHCO 3 20 and Na2HPO 4 1.3) and delivered in a volume of 100 hi. Injections were made using a PicoPump (WPI, New Haven CT). The volume of drug injected into the NTS was carefully monitored by watching the movement of the fluid meniscus in the calibrated glass pipette. For bilateral injections, the drug was initially injected on one side, the pipette withdrawn, repositioned on the contralateral side and the second injection made; thus, injections were made approximately 1 min apart. Some animals received unilateral electrolytic lesions of one NTS 30 min prior to injection of GABA drugs into the contralateral, intact NTS. Lesions were made using a Teflon-insulated tungsten electrode (outside diameter 150/~m) with approximately 150/~m of the tip exposed. The coordinates for creating the lesions were the same as for microinjection. Lesions were made by passing anodal current of 1 mA (Grass LM5, Quincy, MA) for 10 s through the electrode. A clip attached to the neck muscles served as the cathode. Effectiveness of the lesion was confirmed by the presence of a pressor response to NIP (10 nmol) injected into the intact NTS 23; lesioned animals that did not show a pressor response to NIP were discarded from the study. At the conclusion of each experiment, the animal was anesthetized with urethane (1.5 g/kg, i.v.) and perfused intracardially with saline followed by 10% buffered formalin. The brain stem was removed, frozen, and sectioned (40 /~m) using a microtome. Sections were mounted on microscope slides and stained with Cresyl violet. Only animals in which pipette tracks and lesions were centered in the medial subnucleus of the NTS at the level of the area postrema were included in the data analysis. Animals in which there was evidence of bleeding in the NTS were not included in the data analysis. In some experiments, an injection of 100 nl of Fast green dye was made into the NTS prior to sacrifice. Injection and lesion sites were similar to those in previous experiments (see ref. 3). The ability of local administration of 7-vinylGABA (GVG) to inhibit GABA transaminase (GABA-T) was assessed by measuring the accumulation of GABA in the NTS following injection of GVG. GVG or vehicle was injected unilaterally into the NTS and rats were sacrificed 45 or 90 min later, 2 min after the i.v. injection of 3-mercapto-proprionic acid. (3-Mercaptoproprionic acid was used to prevent the rapid postmortem accumulation of GABA25.) The brainstem was removed and frozen. A 1 mm section of brainstem extending rostrally from the caudal tip of the area postrema was cut
E
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Fig. 1. Effects of phaclofen injected into NTS on arterial pressure and heart rate. AP, MAP and HR recordings from a typical chloralose-anesthetized rat with a unilateral NTS lesion following microinjection of PHAC (4.0 nmol) into the intact NTS.
while the brain was still frozen, and the injected NTS was punched from this region using an 0.8 mm diameter needle. Single punches were homogenized in 100/~10.1 M perehloric acid containing 50/~M a-aminobutyric acid (AABA; used as an internal standard) and GABA and GVG levels were measured by fluorometric detection of the o-phthalaldehyde (OPA) derivative, following separation by reverse-phase HPLC. Briefly, 20/A of sample were mixed for 2 min with 40 gl OPA reagent (Pierce Chemical). This mixture was then injected over the HPLC column (Waters gBondapak Radial Compression Cartridge). The mobile phase was 25% acetonitrile in 0.1 M sodium phosphate, pH 5.9, pumped at 3.0 ml/min. After elution of GABA (approx. 7 min), A A B A (approx. 9 min), and GVG (approx. 15 min) the acetonitrile concentration was increased to 50% for 5 min and then the column was reequilibrated with 25% acetonitrile for 10 min before the injection of the next sample. Chromatograms were acquired and analyzed using a computerbased system (Baseline, Dynamic Solutions). Phaclofen was purchased from Tocris Neuramin (Essex, U.K.) and y-vinylGABA was generously donated by MerreI-Dow (Strasbourg, France). Muscimol was purchased from Research Biochemicals (Wayland, MA) and nipecotic acid purchased from Sigma (St. Louis, MO). (-)-Baclofen was generously donated by CibaGeigy (Summit, N J). Data are expressed as means + S.E.M. and were analyzed by analysis of variance followed by the Newman-Keuls Test (PC ANOVA, Human Systems Dynamics, Northridge, CA).
RESULTS U n i l a t e r a l i n j e c t i o n into t h e N T S f o l l o w i n g d e s t r u c t i o n o f t h e c o n t r a l a t e r a l N T S o f P H A C (4 n m o l in 100 nl aCSF) resulted
in a t r a n s i e n t
d e c r e a s e in m e a n
AP
( M A P , - 8 + 2 m m H g ; n = 7; P < 0.05) w i t h little c h a n g e in H R (Fig. 1). S m a l l e r doses o f P H A C (0.5 a n d 2 n m o l ; n = 6 at e a c h d o s e ) had no effect on e i t h e r A P o r H R . L a r g e r doses w e r e not t e s t e d d u e to t h e l i m i t e d solubility of this drug in p h y s i o l o g i c a l solutions. In animals with u n i l a t e r a l lesions o f t h e N T S , i n j e c t i o n of P H A C ( 0 . 5 - 4 n m o l ) into the intact N T S p r o d u c e d a d o s e - d e p e n d e n t d e c r e a s e in t h e p r e s s o r r e s p o n s e elicited
237
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Fig. 2. Phaclofen attenuates the pressor response to baclofen but not the pressor response to muscimol. AP, M A P and H R recordings from typical chloralose-anesthetized rat with unilateral lesion of the NTS and baclofen (upper) or muscimol (lower) injected into the intact NTS, followed approximately 3 rain later by injection of phaclofen into the same site. These tracings are typical of the data included in Table I.
Fig. 3. Phaclofen reverses the pressor response elicted by nipecotic acid. AP, MAP and HR recordings from typical chloraloseanesthetized rat with unilateral lesion of the NTS and nipecotic acid injected into the intact NTS, followed approximately 2 min later by injection of phaclofen or vehicle into the same site.
by prior injection of BAC (20 pmol; the minimum dose to elicit the maximal rise in AP 23) into the NTS (Table I, Fig. 2). PHAC (4 nmol) totally reversed the effect of a
smaller dose of BAC (5 pmol) but was less effective at reversing the response to a higher dose of BAC (100 pmol) (Table II). Injection of PHAC (4 nmol) into the
TABLE I
Effect of phaclofen on the response to GABA agonists injected into the NTS Groups of chloralose-anesthetized rats (n = 5-7) with unilateral lesions of the NTS received an injection of either BAC or MUS into the intact NTS. At the time of the peak pressor response PHAC was injected at the same site as the G A B A agonist. Results are expressed as the maximum change in blood pressure and heart rate following the administration of each drug, and data were analyzed with analysis of variance and the Newman-Keuls test. With the doses used, BAC and MUS elicited pressor responses of the same magnitude, but only B A C produced a significant change in heart rate. PHAC antagonized the response to 20 pmol BAC in a dose-dependent manner, and 4 nmol PHAC was less effective attenuating the pressor response to 100 pmol BAC compared to 20 pmol BAC. PHAC did not significantly effect the response elicited by MUS.
Treatment
Baseline MAP
20 pmol BAC/0.5 nmol PHAC 20 pmol BAC/2.0 nmol PHAC 20 pmol BAC/4.0 nmol PHAC
100 pmol BAC/4.0 nmol PHAC 5 pmol MUS/4.0 nmol PHAC
110+ 110 + 104 + 108 + 106 +
HR 4 5 4 4 1
355 350 370 370 365
+ + + + +
12 15 12 12 10
Change with agonist
Change with PHA C
MAP
HR
MAP
+50+ 2 + 48 + 4 + 50 __ 2 + 54 + 4 +46 + 6
+20+ + 25 + + 17 + +20 + + 10 +
2 2 2 2 6
-2 -10 -24 -14
+ + + +
HR 1 4 2 2
0+ 0 -15 + 5 -17 + 2 -10 + 2
-4 + 2
-2 + 2
238 TABLE II
125"
I
Effect of GVG injected into the NTS on arterial pressure Groups of chloralose-anesthetized rats (n = 5-6) received bilateral injections into the NTS of various doses of GVG. Data are expressed as the maximum change in mean arterial pressure following injection of GVG and the time it took for mean arterial pressure to return to baseline levels following GVG injection. Data were analyzed by analysis of variance and the Newman-Keuls test. Only the highest dose significantly altered heart rate (+ 15 + 3 beats/min).
!'-
2 ~
Dose (nmol)
Baseline MAP (mmHg)
Maximum change in MAP (mmHg)
Duration (rnin)
0 1 5 25 100
106+4 106 + 5 110+ 6 110 + 4 106 + 4
0+2 +2 + 2 + 3 6 + 5 *# +38 + 4* +43 + 2*
0+0 0.6 + 0.6 6.3 + 0.3 *# 8.4 + 0.6 *# 19.2 + 1.0 *#
* Indicates significant difference from the vehicle-treated group, P
100
75
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511
T
I-z
25
_ o
< 0.05. # Indicates significantdifferencefrom the smaller doses, P < 0.05. NTS did not alter the pressor response elicited by injection of MUS (5 pmol; the minimum dose to elicit the maximal rise in AP 3) into the NTS (Table I, Fig. 2). Injection into the NTS of NIP (10 nmol; the minimum dose to elicit the maximal rise in AP 3) increased MAP (+41 + 4 mmHg) and this response was substantially reversed by injection of PHAC (4 nmol) injected during the peak of the pressor response (-35 + 4 mmHg, 87% of response; n = 7; P < 0.005; Fig. 3). Injection of GVG (1-100 nmol) bilaterally into the NTS increased AP, with little effect on HR. This pressor response was dose-dependent with respect to both magnitude and duration (Fig. 4, Table II). Although GVG is reported to be an irreversible inhibitor of GABA-T, the pressor response to GVG injected into the NTS could be elicited with repeated injections; the pressor response to
20C -1E E E
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Fig. 4. Effect o f G V G on arterial pressure and heart rate. AP, M A P
and HR recordings from a typcial chloralose-anesthetized rat following bilateral injection into the NTS of GVG (5 nmol). This recording is typical of the data included in Table II.
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Fig. 5. Effect of GVG on GABA levels in the NTS. GABA levels in the NTS were measured in groups of rats (n = 5) 45 min following the injection into the NTS of 0 (control), 5, 20 or 100 nmol of GVG or 90 min following the injection of 20 nmol of GVG. Data were analysed by analysis of variance followed by the Newman-Keuls test. GVG produced a time-dependent increase in GABA levels in the NTS, with 5-100 nmol not being significantly different from each other (P < 0.05).
bilateral injection into the NTS of GVG (20 or 100 nmol) was similar in magnitude and duration to the response elicited 30 min previously by that dose of the drug (n = 4 for each dose; data not shown). To confirm that these doses of GVG inhibited GABA-T when injected into the NTS, G A B A levels in the NTS were measured in rats sacrificed 0 (vehicle injection), 45, or 90 min following injection of GVG. Injection of 20 nmol of GVG into the NTS caused a linear increase in G A B A levels (Fig. 5). Injection of 5 or 100 nmol of G V G into the NTS elicited increases in NTS G A B A content similar to the 20 nmol dose of GVG. The level of G V G in the NTS measured 45 min following injection of the 20 nmol dose was 57.1 _+ 6.0 pmol/#g protein. In 6 rats, PHAC (4 nmol) was able to reverse the pressor response elicited by injection into the NTS of GVG (25 nmol; Fig. 6). Injection of GVG unilaterally into the NTS following destruction of the contralateral NTS elicited an increase in MAP of 38 + 6 mmHg and PHAC (4 nmol) then decreased MAP 36 + 6 mmHg immediately upon administration into the NTS. However, this was not a consistent finding; in an additional 4
239
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4 nmol
20o[ 160
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Fig. 6. Phaclofen reverses the pressor response elicited by GVG. AP, MAP and HR recordings from typical chloralose-anesthetized rats with unilateral lesions of the NTS and GVG injected into the intact NTS, followed approximately 2 min later by injection of phaclofen or vehicle into the same site.
rats treated in an identical manner, PHAC appeared to have no effect on the pressor response evoked by GVG injection. DISCUSSION NIP injected into the NTS of chloralose-anesthetized rats increases blood pressure 3. Since NIP is a selective inhibitor of G A B A uptake into neurons and glia5'15-18, the simplest explanation of this observation is that NIP injected into the NTS increases AP by enhancing the action of G A B A normally present in synapses in the NTS. In the present study, we examined the effects of a member of a second class of indirect-acting G A B A agonists, drugs that inhibit G A B A metabolism. Injections into the NTS of GVG, an irreversible inhibitor of G A B A - T 11'19'2°, elicited a dose-dependent increase in AP. However, following GVG injection into the NTS the inhibition of GABA-T, as reflected by an increase in G A B A levels, lasts for hours and is not consistent with the pressor response lasting less than 20 min. Further-
more, following a dose of G V G that maximally inhibits GABA-T, the pressor response to G V G can still be elicited by a second injection of GVG. Thus, it appears unlikely that GVG elicits a pressor response by inhibiting GABA-T. Since G V G is also reported to competetively inhibit GABA uptake in the same dose range as it inhibits GABA-T 2°, this mechanism appears to better explain the GVG evoked pressor response. Based on the levels of GVG that were measured in the NTS, the GVG concentration in the NTS 45 min after injection of 20 nmol can be estimated to be approximately 5 mM. (IC5o values for GVG inhibition of G A B A - T and G A B A uptake are 350/tM and l l 0 p M , respectively2°.) A second GABA-T inhibitior, gabaculline, has also been examined; this agent also elicits an increase in AP when injected into the NTS (A. Sved, unpublished results). As with GVG, gabaculline's action appears not to be mediated through inhibition of GABA-T, and may result from inhibition of G A B A uptake 2°. It is noteworthy that although GVG (and gabaculline) markedly increase the level of G A B A in the NTS, subsequent injections of GVG, gabaculline, or nipecotic acid do not result in enhanced pressor responses (present report and A. Sved, unpublished results). These results suggest that following inhibition of GABA-T there is no increase in synaptic G A B A concentration although tissue levels do increase, a suggestion that has been made previously (see ref. 10). Since injection into the NTS of either direct-acting G A B A A or GABAB agonists causes an increase in AP 3'23, indirect-acting G A B A agonists such as NIP and GVG may elicit a pressor response through stimulation of either G A B A A or G A B A B receptors. We previously reported 23 that BIC, a specific G A B A A antagonist 4'9, did not attenuate the pressor response produced by injection of NIP into the NTS, suggesting that the pressor response elicited by NIP was mediated via G A B A B receptors. The present studies demonstrate that PHAC, a selective G A B A B antagonist 8'13"14, does block the pressor reponse to NIP (and another indirect-acting agonist, GVG), providing direct evidence is support of our previous suggestion. These results indicate that potentiating the action of endogenous G A B A in the NTS leads to an increase in AP by stimulating G A B A B receptors. (It is unclear at present why PHAC was ineffective at attenuating the response to GVG in several animals.) G A B A uptake blocking agents, such as NIP, should act by enhancing the action of G A B A present in the synapse. If G A B A in synapses in the NTS normally acts on G A B A B receptors, and this action leads to increased AP, then it would be expected that blockade of G A B A B receptors would result in a decrease in AP. Such a decrease in AP was observed following injection into the NTS of the highest dose of PHAC tested, 4 nmol.
240 Although this dose of P H A C decreased A P by only 8 + 2 m m H g , higher doses of the drug were not tested due to its limited solubility in physiological solutions. It should
of G A B A B but not G A B A A receptors. These results indicate that G A B A , e n d o g e n o u s l y released in the NTS, acts on G A B A B receptors to increase AP.
be n o t e d that the 4 nmol dose of P H A C was not able to completely block the pressor reponse elicited by B A C , suggesting that 4 nmol is not the maximally effective dose of this drug. In conclusion, these studies demonstrate that the increase in A P elicited by injection into the NTS of indirectly acting G A B A agonists is inhibited by blockade
REFERENCES 1 Bousquet, P., Feldman, J., Bloch, R. and Schwartz, J., Evidence for a neuromodulatory role of GABA at the first synapse of the baroreceptor reflex pathway. Effects of GABA derivatives injected into the NTS, Naunyn-Schmiedeberg's Arch. Pharmacol., 319 (1982) 168-171. 2 Castro, R. and Phillips, M.I., Mechanism of pressor effects by angiotensin in the nucleus tractus solitarius, Am. J. Physiol., 247 (1984) R575-R581. 3 Catelli, J.M., Giakas, W.J. and Sved, A.E, GABAergic mechanisms in the NTS alter blood pressure and vasopressin release, Brain Research, 403 (1987) 279-289. 4 Curtis, D.R., Duggan, A.W., Felix, D. and Johnston, G.A.R., Bicuculline an antagonist of GABA and synaptic inhibition in the spinal cord of the cat, Brain Research, 32 (1971) 69-96. 5 Dalkara, T., Nipecotic acid, an uptake blocker, prevents fading of the ),-aminobutyric acid effect, Brain Research, 366 (1986) 314-319. 6 DeJong, W. and Nijkamp, P.E, Centrally induced hypertension and bradycardia after administration of a-methylnoradrenaline into the area of the nucleus tractus solitarii of the cat, Br. J. Pharmacol., 58 (1976) 593-598. 7 Doba, N. and Reis, D.J., Acute fulminating neurogenic hypertension produced by brain stem lesion in the rat, Circ. Res., 32 (1973) 584-593. 8 Dutar, P. and Nicoll, R.A., A physiological role for GABAB receptors in the central nervous system, Nature, 332 (1988) 156-158. 9 Enna, S.J. and Snyder, S.H., Properties of y-aminobutyric acid (GABA) receptor binding in rat brain synaptic membrane fraction, Brain Research, 100 (1975) 81-97. 10 Iadarola, M.J. and Gale, K., Cellular compartments of GABA in brain and their relationship to anticonvulsant activity, Mol. Cell. Biochem., 39 (1981) 305-330. 11 Jung, M.J., Lippert, B., Metcalf, B.W., Bohlen, P. and Schechter, P.J., Gamma-vinyl GABA (4-amino-hex-5-enoic acid), a new selective irreversible inhibitor of GABA-T: effects on brain GABA metabolism in mice, J. Neurochem., 29 (1977) 787-802. 12 Kalia, M.P., Location of aortic and carotid baroreceptor and chemoreceptor primary afferents in the brain stem. In J.P. Buckley and C.M. Ferrario (Eds.), Central Nervous System Mechanisms in Hypertension, Raven Press, New York, 1981, pp. 9-24.
Acknowledgements. These studies were supported by a grant from the National Institutes of Health (HL-38786). Alan F. Sved was supported by an Established Investigator Award from the American Heart Association. Technical assistance was provided by Michele Flasher, Beth Ann Marne, and Jesse Salter; their work on this project is greatly appreciated.
13 Kerr, D.I., Ong, J., Prager, R.H., Gynther, B.D. and Curtis, D.R., Phaclofen: a peripheral and central baclofen antagonist, Brain Research, 405 (1987) 150-154. 14 Kerr, D.I., Ong, J., Johnston, G.A.R., Abbenante, J. and Prager, R.H., 2-Hydroxy-saclofen: an improved antagonist at central and peripheral GABAB receptors, Neurosci. Lea., 92 (1988) 92-96. 15 Korn, S.J. and Dingledine, R., Inhibition of GABA uptake in the rat hippocampal slice, Brain Research, 368 (1986) 247-255. 16 Krogsgaard-Larsen, P., Inhibitors of the GABA uptake systems, Mol. Cell. Biochem., 31 (1980) 105-121. 17 Krogsgaard-Larsen, P., y-Aminobutyric acid agonists; antagonists and uptake inhibitors: Design and therapeutic aspects, J. Med. Chem., 24 (1981) 1377-1383. 18 Krogsgaard-Larsen, P. and Johnston, G.A.R., Inhibition of GABA uptake in rat brain slices by nipecotic acid, various isoxazoles and related compounds, J. Neurochem., 25 (1975) 797-802. 19 Lippert, B., Metcalf, B.W., Jung, M.J. and Casara, P., 4Amino-hex-5-enoic acid, a selective catalytic inhibitor of 4aminobutyric acid aminotransferase in mammalian brain, Eur. J. Biochem., 74 (1977) 441-445. 20 Loscher, W., Effects of inhibitors of GABA transaminase on the synthesis, binding, uptake, and metabolism of GABA, J. Neurochem., 34 (1980) 1603-1608. 21 Matsuguchi, H., Sharabi, EM., Gordon, EJ., Johnston, A.K. and Schmid, P.G., Blood pressure and heart rate responses to microinjection of vasopressin into the nucleus tractus solitarius region of the rat, Neuropharmacology, 21 (1982) 687-693. 22 Reis, D.J., The nucleus tractus solitarii (NTS) and experimental neurogenic hypertension. In M.J. Hughes and C.D. Barnes (Eds.), Neural Control of Circulation, Academic Press, New York, 1980, pp. 81-102. 23 Sved, J. C. and Sved, A.E, Cardiovascular responses elicited by ~,-aminobutyricacid in the nucleus tractus solitarius: evidence for action at the GABAB receptor, Neuropharmacology, 28 (1989) 515-520. 24 Talman, W.T., Granata, A.R. and Reis, D.J., Glutamatergie mechanisms in the nucleus tractus solitarius in blood pressure control, Fed. Proc., 43 (1984) 39-44. 25 Van der Heyden, J.A.M. and Korf, J., Regional levels of GABA in the brain: rapid semiautomated assay and prevention of postmortem increase by 3-mercapto-proprionic acid, J. Neurochem., 31 (1978) 197-203.
235
BRES 15809
Endogenous G A B A acts on GABAB receptors in nucleus tractus solitarius to increase blood pressure Alan E Sved and Judith C. Sved Department of Behavioral Neuroscience, University of Pittsburgh, Pittsburgh, PA 15260 (U.S.A.) (Accepted 6 March 1990) Key words: Phaclofen; Nipecotic acid; 7-Aminobutyric acid transaminase inhibitor; Baclofen; Hypertension
Previous studies found that injection of the G A B A uptake inhibitor nipecotic acid into the nucleus tractus solitarius (NTS) increases arterial pressure. This effect of nipecotic acid was not antagonized by the selective G A B A A receptor blocking agent bicuculline, suggesting that the action of nipecotic acid was mediated through an action of G A B A on G A B A B receptors in the NTS. The present studies examined this issue using a newly described G A B A s antagonist, phaclofen. Injection of phaclofen (4 nmol in 100 nl artificial CSF) into the NTS of chloralose-anesthetized rats produced a slight decrease in arterial pressure (-8 + 2 mmHg) lasting less than 1 min. Smaller doses had no effect. Phaclofen antagonized in a dose-dependent (0.5-4 nmol) manner the increase in arterial pressure produced by injection into the NTS of the G A B A n agonist baclofen (5-100 pmol). In contrast, phaclofen had no effect on the pressor response elicited by injection into the NTS of the G A B A A agonist muscimol. Phaclofen (4 nmol) injected into the NTS totally reversed the increase in blood pressure elicited by injection into the NTS of a maximally effective dose of nipecotic acid (10 nmol). Phaclofen also inhibited the pressor response elicited by injection into the NTS of another indirectly acting G A B A agonist, ?-vinylGABA (GVG). Although GVG is an effective inhibitor of G A B A transaminase, the enzyme involved in the metabolism of GABA, the time course of inhibition of G A B A transaminase evoked by GVG was not consistent with the pressor response being produced by this mechanism. However, the pressor response elicited by GVG is consistent with its reported ability to inhibit G A B A uptake. These results support the hypothesis that G A B A tonically released in the NTS acts specifically on G A B A n receptors to increase blood pressure.
INTRODUCTION The nucleus tractus solitarius (NTS), the site of termination within the brain of baroreceptor afferent fibers 12, has repeatedly been shown to be involved in the regulation of arterial pressure (AP) 22. Many neuroactive substances present within the NTS have been shown to be involved in this process (e.g. refs. 1, 2, 3, 6, 21 and 24), although the manner in which any specific neurotransmitter acts physiologically in the regulation of AP has not been completely elucidated. We have previously demonstrated that injection into the NTS of nipecotic acid (NIP), a selective inhibitor of G A B A uptake 5'15-18, produces an increase in A P 3'23. The most straight-forward interpretation of this finding is that potentiating the action of synaptically released G A B A in the NTS, by blocking its clearance from the synapse by uptake, elicits an increase in AP. Since selective stimulation of either G A B A A or G A B A B receptors in the NTS can elicit a pressor response 3'23, G A B A tonically released into synapses in the NTS may normally act on either or both of these receptors. Recent studies 23 indicate that the pressor response evoked by injection of
NIP into the NTS is not attenuated by the selective G A B A A antagonist bicuculline (BIC), suggesting that G A B A may normally act on G A B A B receptors in the NTS to maintain AP. At the time those studies were conducted, no selective G A B A a antagonists were available. However, a series of baclofen derivatives that act as selective antagonists of the G A B A B receptor have recently been described 13'14, with the phosphonic acid derivative of baclofen (BAC), phaclofen (PHAC) being the prototypic agent. The present studies examined the effect of administering PHAC into the NTS on baseline AP and the increase in AP elicited by stimulation of G A B A receptors in the NTS. The results support our previous suggestion that potentiating the action of tonically released G A B A in the NTS elevates AP through stimulation of G A B A B receptors. MATERIALS AND METHODS Adult male Sprague-Dawley rats, 300-450 g (Zivic-Miller, Allison Park, PA) were housed in single cages in a temperaturecontrolled room on a 12 h light/dark cycle with food and tap water available ad libitum for at least 1 week prior to use in experiments.
Correspondence: A. Sved, Department of Behavioral Neuroscience, University of Pittsburgh, 446 Crawford Hall, Pittsburgh, PA 15260, U.S.A. D~fi-ROO~/O~/lflq ~if)(~ IOCKIl=;l~vi~r ~eienr~e Pnhli~her~ B V (Riomedica! r)ivision)
236 Rats were anesthetized with halothane (2% in 100% 02 administered through a nose cone). A cannula (PE 50 tubing filled with heparinized saline) was inserted in the right femoral artery for recording arterial pressure (AP), mean arterial pressure (MAP) and heart rate (HR) (Grass Model 7 Physiograph). A second cannula was placed in the right femoral vein for administering drugs. The trachea was cannulated and rats artificially ventilated with 2% halothane in 100% 0 2 (Harvard Small Animal Respirator) following the administration of a muscle relaxant (d-tubocurarine, 0.5 mg/kg, i.v., supplemented hourly with 0.2 mg/kg, i.v.). Rats were placed in a stereotaxic instrument (Kopf) with the incisor bar positioned 11 mm below the interaural line. The dorsal surface of the brain stem was exposed by limited craniotomy and, with the aid of a surgical microscope, the area postrema visualized. a-Chloralose was administered (60 mg/kg, i.v., supplemented hourly with 20 mg/kg, i.v.) and the halothane terminated. Rats were ventilated with 100% O 2 throughout the remainder of the experiment. Blood gases were measured (IL model 1304 blood gas analyzer) periodically and the respirator was adjusted to maintain arterial pCO 2 in the range of 38-45 mm Hg. Injections of GABA agents were made into the NTS using single or double barrel glass micropipettes pulled to an outer diameter of 40-50/~m. Double barrel pipettes were used in all experiments in which an agonist and antagonist were injected into the same site. The tip of the micropipette was positioned 0.5 mm rostral to the calamus scriptorius, 0.5 mm lateral from the midline and 0.5 mm deep to the surface of the brain stem for injection into the NTS. All drugs were dissolved in artificial cerebrospinal fluid (aCSF (in mM): NaCi 128, KCI 2.6, CaC! 2 1.3, MgCl 2 0.9, NaHCO 3 20 and Na2HPO 4 1.3) and delivered in a volume of 100 hi. Injections were made using a PicoPump (WPI, New Haven CT). The volume of drug injected into the NTS was carefully monitored by watching the movement of the fluid meniscus in the calibrated glass pipette. For bilateral injections, the drug was initially injected on one side, the pipette withdrawn, repositioned on the contralateral side and the second injection made; thus, injections were made approximately 1 min apart. Some animals received unilateral electrolytic lesions of one NTS 30 min prior to injection of GABA drugs into the contralateral, intact NTS. Lesions were made using a Teflon-insulated tungsten electrode (outside diameter 150/~m) with approximately 150/~m of the tip exposed. The coordinates for creating the lesions were the same as for microinjection. Lesions were made by passing anodal current of 1 mA (Grass LM5, Quincy, MA) for 10 s through the electrode. A clip attached to the neck muscles served as the cathode. Effectiveness of the lesion was confirmed by the presence of a pressor response to NIP (10 nmol) injected into the intact NTS 23; lesioned animals that did not show a pressor response to NIP were discarded from the study. At the conclusion of each experiment, the animal was anesthetized with urethane (1.5 g/kg, i.v.) and perfused intracardially with saline followed by 10% buffered formalin. The brain stem was removed, frozen, and sectioned (40 /~m) using a microtome. Sections were mounted on microscope slides and stained with Cresyl violet. Only animals in which pipette tracks and lesions were centered in the medial subnucleus of the NTS at the level of the area postrema were included in the data analysis. Animals in which there was evidence of bleeding in the NTS were not included in the data analysis. In some experiments, an injection of 100 nl of Fast green dye was made into the NTS prior to sacrifice. Injection and lesion sites were similar to those in previous experiments (see ref. 3). The ability of local administration of 7-vinylGABA (GVG) to inhibit GABA transaminase (GABA-T) was assessed by measuring the accumulation of GABA in the NTS following injection of GVG. GVG or vehicle was injected unilaterally into the NTS and rats were sacrificed 45 or 90 min later, 2 min after the i.v. injection of 3-mercapto-proprionic acid. (3-Mercaptoproprionic acid was used to prevent the rapid postmortem accumulation of GABA25.) The brainstem was removed and frozen. A 1 mm section of brainstem extending rostrally from the caudal tip of the area postrema was cut
E
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300~
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Fig. 1. Effects of phaclofen injected into NTS on arterial pressure and heart rate. AP, MAP and HR recordings from a typical chloralose-anesthetized rat with a unilateral NTS lesion following microinjection of PHAC (4.0 nmol) into the intact NTS.
while the brain was still frozen, and the injected NTS was punched from this region using an 0.8 mm diameter needle. Single punches were homogenized in 100/~10.1 M perehloric acid containing 50/~M a-aminobutyric acid (AABA; used as an internal standard) and GABA and GVG levels were measured by fluorometric detection of the o-phthalaldehyde (OPA) derivative, following separation by reverse-phase HPLC. Briefly, 20/A of sample were mixed for 2 min with 40 gl OPA reagent (Pierce Chemical). This mixture was then injected over the HPLC column (Waters gBondapak Radial Compression Cartridge). The mobile phase was 25% acetonitrile in 0.1 M sodium phosphate, pH 5.9, pumped at 3.0 ml/min. After elution of GABA (approx. 7 min), A A B A (approx. 9 min), and GVG (approx. 15 min) the acetonitrile concentration was increased to 50% for 5 min and then the column was reequilibrated with 25% acetonitrile for 10 min before the injection of the next sample. Chromatograms were acquired and analyzed using a computerbased system (Baseline, Dynamic Solutions). Phaclofen was purchased from Tocris Neuramin (Essex, U.K.) and y-vinylGABA was generously donated by MerreI-Dow (Strasbourg, France). Muscimol was purchased from Research Biochemicals (Wayland, MA) and nipecotic acid purchased from Sigma (St. Louis, MO). (-)-Baclofen was generously donated by CibaGeigy (Summit, N J). Data are expressed as means + S.E.M. and were analyzed by analysis of variance followed by the Newman-Keuls Test (PC ANOVA, Human Systems Dynamics, Northridge, CA).
RESULTS U n i l a t e r a l i n j e c t i o n into t h e N T S f o l l o w i n g d e s t r u c t i o n o f t h e c o n t r a l a t e r a l N T S o f P H A C (4 n m o l in 100 nl aCSF) resulted
in a t r a n s i e n t
d e c r e a s e in m e a n
AP
( M A P , - 8 + 2 m m H g ; n = 7; P < 0.05) w i t h little c h a n g e in H R (Fig. 1). S m a l l e r doses o f P H A C (0.5 a n d 2 n m o l ; n = 6 at e a c h d o s e ) had no effect on e i t h e r A P o r H R . L a r g e r doses w e r e not t e s t e d d u e to t h e l i m i t e d solubility of this drug in p h y s i o l o g i c a l solutions. In animals with u n i l a t e r a l lesions o f t h e N T S , i n j e c t i o n of P H A C ( 0 . 5 - 4 n m o l ) into the intact N T S p r o d u c e d a d o s e - d e p e n d e n t d e c r e a s e in t h e p r e s s o r r e s p o n s e elicited
237
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4 nmol
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2°ct
160 ~.
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4 nmol
~aCSF
Fig. 2. Phaclofen attenuates the pressor response to baclofen but not the pressor response to muscimol. AP, M A P and H R recordings from typical chloralose-anesthetized rat with unilateral lesion of the NTS and baclofen (upper) or muscimol (lower) injected into the intact NTS, followed approximately 3 rain later by injection of phaclofen into the same site. These tracings are typical of the data included in Table I.
Fig. 3. Phaclofen reverses the pressor response elicted by nipecotic acid. AP, MAP and HR recordings from typical chloraloseanesthetized rat with unilateral lesion of the NTS and nipecotic acid injected into the intact NTS, followed approximately 2 min later by injection of phaclofen or vehicle into the same site.
by prior injection of BAC (20 pmol; the minimum dose to elicit the maximal rise in AP 23) into the NTS (Table I, Fig. 2). PHAC (4 nmol) totally reversed the effect of a
smaller dose of BAC (5 pmol) but was less effective at reversing the response to a higher dose of BAC (100 pmol) (Table II). Injection of PHAC (4 nmol) into the
TABLE I
Effect of phaclofen on the response to GABA agonists injected into the NTS Groups of chloralose-anesthetized rats (n = 5-7) with unilateral lesions of the NTS received an injection of either BAC or MUS into the intact NTS. At the time of the peak pressor response PHAC was injected at the same site as the G A B A agonist. Results are expressed as the maximum change in blood pressure and heart rate following the administration of each drug, and data were analyzed with analysis of variance and the Newman-Keuls test. With the doses used, BAC and MUS elicited pressor responses of the same magnitude, but only B A C produced a significant change in heart rate. PHAC antagonized the response to 20 pmol BAC in a dose-dependent manner, and 4 nmol PHAC was less effective attenuating the pressor response to 100 pmol BAC compared to 20 pmol BAC. PHAC did not significantly effect the response elicited by MUS.
Treatment
Baseline MAP
20 pmol BAC/0.5 nmol PHAC 20 pmol BAC/2.0 nmol PHAC 20 pmol BAC/4.0 nmol PHAC
100 pmol BAC/4.0 nmol PHAC 5 pmol MUS/4.0 nmol PHAC
110+ 110 + 104 + 108 + 106 +
HR 4 5 4 4 1
355 350 370 370 365
+ + + + +
12 15 12 12 10
Change with agonist
Change with PHA C
MAP
HR
MAP
+50+ 2 + 48 + 4 + 50 __ 2 + 54 + 4 +46 + 6
+20+ + 25 + + 17 + +20 + + 10 +
2 2 2 2 6
-2 -10 -24 -14
+ + + +
HR 1 4 2 2
0+ 0 -15 + 5 -17 + 2 -10 + 2
-4 + 2
-2 + 2
238 TABLE II
125"
I
Effect of GVG injected into the NTS on arterial pressure Groups of chloralose-anesthetized rats (n = 5-6) received bilateral injections into the NTS of various doses of GVG. Data are expressed as the maximum change in mean arterial pressure following injection of GVG and the time it took for mean arterial pressure to return to baseline levels following GVG injection. Data were analyzed by analysis of variance and the Newman-Keuls test. Only the highest dose significantly altered heart rate (+ 15 + 3 beats/min).
!'-
2 ~
Dose (nmol)
Baseline MAP (mmHg)
Maximum change in MAP (mmHg)
Duration (rnin)
0 1 5 25 100
106+4 106 + 5 110+ 6 110 + 4 106 + 4
0+2 +2 + 2 + 3 6 + 5 *# +38 + 4* +43 + 2*
0+0 0.6 + 0.6 6.3 + 0.3 *# 8.4 + 0.6 *# 19.2 + 1.0 *#
* Indicates significant difference from the vehicle-treated group, P
100
75
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511
T
I-z
25
_ o
< 0.05. # Indicates significantdifferencefrom the smaller doses, P < 0.05. NTS did not alter the pressor response elicited by injection of MUS (5 pmol; the minimum dose to elicit the maximal rise in AP 3) into the NTS (Table I, Fig. 2). Injection into the NTS of NIP (10 nmol; the minimum dose to elicit the maximal rise in AP 3) increased MAP (+41 + 4 mmHg) and this response was substantially reversed by injection of PHAC (4 nmol) injected during the peak of the pressor response (-35 + 4 mmHg, 87% of response; n = 7; P < 0.005; Fig. 3). Injection of GVG (1-100 nmol) bilaterally into the NTS increased AP, with little effect on HR. This pressor response was dose-dependent with respect to both magnitude and duration (Fig. 4, Table II). Although GVG is reported to be an irreversible inhibitor of GABA-T, the pressor response to GVG injected into the NTS could be elicited with repeated injections; the pressor response to
20C -1E E E
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Fig. 4. Effect o f G V G on arterial pressure and heart rate. AP, M A P
and HR recordings from a typcial chloralose-anesthetized rat following bilateral injection into the NTS of GVG (5 nmol). This recording is typical of the data included in Table II.
E
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Fig. 5. Effect of GVG on GABA levels in the NTS. GABA levels in the NTS were measured in groups of rats (n = 5) 45 min following the injection into the NTS of 0 (control), 5, 20 or 100 nmol of GVG or 90 min following the injection of 20 nmol of GVG. Data were analysed by analysis of variance followed by the Newman-Keuls test. GVG produced a time-dependent increase in GABA levels in the NTS, with 5-100 nmol not being significantly different from each other (P < 0.05).
bilateral injection into the NTS of GVG (20 or 100 nmol) was similar in magnitude and duration to the response elicited 30 min previously by that dose of the drug (n = 4 for each dose; data not shown). To confirm that these doses of GVG inhibited GABA-T when injected into the NTS, G A B A levels in the NTS were measured in rats sacrificed 0 (vehicle injection), 45, or 90 min following injection of GVG. Injection of 20 nmol of GVG into the NTS caused a linear increase in G A B A levels (Fig. 5). Injection of 5 or 100 nmol of G V G into the NTS elicited increases in NTS G A B A content similar to the 20 nmol dose of GVG. The level of G V G in the NTS measured 45 min following injection of the 20 nmol dose was 57.1 _+ 6.0 pmol/#g protein. In 6 rats, PHAC (4 nmol) was able to reverse the pressor response elicited by injection into the NTS of GVG (25 nmol; Fig. 6). Injection of GVG unilaterally into the NTS following destruction of the contralateral NTS elicited an increase in MAP of 38 + 6 mmHg and PHAC (4 nmol) then decreased MAP 36 + 6 mmHg immediately upon administration into the NTS. However, this was not a consistent finding; in an additional 4
239
120[
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160
~ 120 80
~" 3OOl25 nmol
t ,c
4 nmol
20o[ 160
~2oI 801" "~
160
~ 120 80
25 nmol
Fig. 6. Phaclofen reverses the pressor response elicited by GVG. AP, MAP and HR recordings from typical chloralose-anesthetized rats with unilateral lesions of the NTS and GVG injected into the intact NTS, followed approximately 2 min later by injection of phaclofen or vehicle into the same site.
rats treated in an identical manner, PHAC appeared to have no effect on the pressor response evoked by GVG injection. DISCUSSION NIP injected into the NTS of chloralose-anesthetized rats increases blood pressure 3. Since NIP is a selective inhibitor of G A B A uptake into neurons and glia5'15-18, the simplest explanation of this observation is that NIP injected into the NTS increases AP by enhancing the action of G A B A normally present in synapses in the NTS. In the present study, we examined the effects of a member of a second class of indirect-acting G A B A agonists, drugs that inhibit G A B A metabolism. Injections into the NTS of GVG, an irreversible inhibitor of G A B A - T 11'19'2°, elicited a dose-dependent increase in AP. However, following GVG injection into the NTS the inhibition of GABA-T, as reflected by an increase in G A B A levels, lasts for hours and is not consistent with the pressor response lasting less than 20 min. Further-
more, following a dose of G V G that maximally inhibits GABA-T, the pressor response to G V G can still be elicited by a second injection of GVG. Thus, it appears unlikely that GVG elicits a pressor response by inhibiting GABA-T. Since G V G is also reported to competetively inhibit GABA uptake in the same dose range as it inhibits GABA-T 2°, this mechanism appears to better explain the GVG evoked pressor response. Based on the levels of GVG that were measured in the NTS, the GVG concentration in the NTS 45 min after injection of 20 nmol can be estimated to be approximately 5 mM. (IC5o values for GVG inhibition of G A B A - T and G A B A uptake are 350/tM and l l 0 p M , respectively2°.) A second GABA-T inhibitior, gabaculline, has also been examined; this agent also elicits an increase in AP when injected into the NTS (A. Sved, unpublished results). As with GVG, gabaculline's action appears not to be mediated through inhibition of GABA-T, and may result from inhibition of G A B A uptake 2°. It is noteworthy that although GVG (and gabaculline) markedly increase the level of G A B A in the NTS, subsequent injections of GVG, gabaculline, or nipecotic acid do not result in enhanced pressor responses (present report and A. Sved, unpublished results). These results suggest that following inhibition of GABA-T there is no increase in synaptic G A B A concentration although tissue levels do increase, a suggestion that has been made previously (see ref. 10). Since injection into the NTS of either direct-acting G A B A A or GABAB agonists causes an increase in AP 3'23, indirect-acting G A B A agonists such as NIP and GVG may elicit a pressor response through stimulation of either G A B A A or G A B A B receptors. We previously reported 23 that BIC, a specific G A B A A antagonist 4'9, did not attenuate the pressor response produced by injection of NIP into the NTS, suggesting that the pressor response elicited by NIP was mediated via G A B A B receptors. The present studies demonstrate that PHAC, a selective G A B A B antagonist 8'13"14, does block the pressor reponse to NIP (and another indirect-acting agonist, GVG), providing direct evidence is support of our previous suggestion. These results indicate that potentiating the action of endogenous G A B A in the NTS leads to an increase in AP by stimulating G A B A B receptors. (It is unclear at present why PHAC was ineffective at attenuating the response to GVG in several animals.) G A B A uptake blocking agents, such as NIP, should act by enhancing the action of G A B A present in the synapse. If G A B A in synapses in the NTS normally acts on G A B A B receptors, and this action leads to increased AP, then it would be expected that blockade of G A B A B receptors would result in a decrease in AP. Such a decrease in AP was observed following injection into the NTS of the highest dose of PHAC tested, 4 nmol.
240 Although this dose of P H A C decreased A P by only 8 + 2 m m H g , higher doses of the drug were not tested due to its limited solubility in physiological solutions. It should
of G A B A B but not G A B A A receptors. These results indicate that G A B A , e n d o g e n o u s l y released in the NTS, acts on G A B A B receptors to increase AP.
be n o t e d that the 4 nmol dose of P H A C was not able to completely block the pressor reponse elicited by B A C , suggesting that 4 nmol is not the maximally effective dose of this drug. In conclusion, these studies demonstrate that the increase in A P elicited by injection into the NTS of indirectly acting G A B A agonists is inhibited by blockade
REFERENCES 1 Bousquet, P., Feldman, J., Bloch, R. and Schwartz, J., Evidence for a neuromodulatory role of GABA at the first synapse of the baroreceptor reflex pathway. Effects of GABA derivatives injected into the NTS, Naunyn-Schmiedeberg's Arch. Pharmacol., 319 (1982) 168-171. 2 Castro, R. and Phillips, M.I., Mechanism of pressor effects by angiotensin in the nucleus tractus solitarius, Am. J. Physiol., 247 (1984) R575-R581. 3 Catelli, J.M., Giakas, W.J. and Sved, A.E, GABAergic mechanisms in the NTS alter blood pressure and vasopressin release, Brain Research, 403 (1987) 279-289. 4 Curtis, D.R., Duggan, A.W., Felix, D. and Johnston, G.A.R., Bicuculline an antagonist of GABA and synaptic inhibition in the spinal cord of the cat, Brain Research, 32 (1971) 69-96. 5 Dalkara, T., Nipecotic acid, an uptake blocker, prevents fading of the ),-aminobutyric acid effect, Brain Research, 366 (1986) 314-319. 6 DeJong, W. and Nijkamp, P.E, Centrally induced hypertension and bradycardia after administration of a-methylnoradrenaline into the area of the nucleus tractus solitarii of the cat, Br. J. Pharmacol., 58 (1976) 593-598. 7 Doba, N. and Reis, D.J., Acute fulminating neurogenic hypertension produced by brain stem lesion in the rat, Circ. Res., 32 (1973) 584-593. 8 Dutar, P. and Nicoll, R.A., A physiological role for GABAB receptors in the central nervous system, Nature, 332 (1988) 156-158. 9 Enna, S.J. and Snyder, S.H., Properties of y-aminobutyric acid (GABA) receptor binding in rat brain synaptic membrane fraction, Brain Research, 100 (1975) 81-97. 10 Iadarola, M.J. and Gale, K., Cellular compartments of GABA in brain and their relationship to anticonvulsant activity, Mol. Cell. Biochem., 39 (1981) 305-330. 11 Jung, M.J., Lippert, B., Metcalf, B.W., Bohlen, P. and Schechter, P.J., Gamma-vinyl GABA (4-amino-hex-5-enoic acid), a new selective irreversible inhibitor of GABA-T: effects on brain GABA metabolism in mice, J. Neurochem., 29 (1977) 787-802. 12 Kalia, M.P., Location of aortic and carotid baroreceptor and chemoreceptor primary afferents in the brain stem. In J.P. Buckley and C.M. Ferrario (Eds.), Central Nervous System Mechanisms in Hypertension, Raven Press, New York, 1981, pp. 9-24.
Acknowledgements. These studies were supported by a grant from the National Institutes of Health (HL-38786). Alan F. Sved was supported by an Established Investigator Award from the American Heart Association. Technical assistance was provided by Michele Flasher, Beth Ann Marne, and Jesse Salter; their work on this project is greatly appreciated.
13 Kerr, D.I., Ong, J., Prager, R.H., Gynther, B.D. and Curtis, D.R., Phaclofen: a peripheral and central baclofen antagonist, Brain Research, 405 (1987) 150-154. 14 Kerr, D.I., Ong, J., Johnston, G.A.R., Abbenante, J. and Prager, R.H., 2-Hydroxy-saclofen: an improved antagonist at central and peripheral GABAB receptors, Neurosci. Lea., 92 (1988) 92-96. 15 Korn, S.J. and Dingledine, R., Inhibition of GABA uptake in the rat hippocampal slice, Brain Research, 368 (1986) 247-255. 16 Krogsgaard-Larsen, P., Inhibitors of the GABA uptake systems, Mol. Cell. Biochem., 31 (1980) 105-121. 17 Krogsgaard-Larsen, P., y-Aminobutyric acid agonists; antagonists and uptake inhibitors: Design and therapeutic aspects, J. Med. Chem., 24 (1981) 1377-1383. 18 Krogsgaard-Larsen, P. and Johnston, G.A.R., Inhibition of GABA uptake in rat brain slices by nipecotic acid, various isoxazoles and related compounds, J. Neurochem., 25 (1975) 797-802. 19 Lippert, B., Metcalf, B.W., Jung, M.J. and Casara, P., 4Amino-hex-5-enoic acid, a selective catalytic inhibitor of 4aminobutyric acid aminotransferase in mammalian brain, Eur. J. Biochem., 74 (1977) 441-445. 20 Loscher, W., Effects of inhibitors of GABA transaminase on the synthesis, binding, uptake, and metabolism of GABA, J. Neurochem., 34 (1980) 1603-1608. 21 Matsuguchi, H., Sharabi, EM., Gordon, EJ., Johnston, A.K. and Schmid, P.G., Blood pressure and heart rate responses to microinjection of vasopressin into the nucleus tractus solitarius region of the rat, Neuropharmacology, 21 (1982) 687-693. 22 Reis, D.J., The nucleus tractus solitarii (NTS) and experimental neurogenic hypertension. In M.J. Hughes and C.D. Barnes (Eds.), Neural Control of Circulation, Academic Press, New York, 1980, pp. 81-102. 23 Sved, J. C. and Sved, A.E, Cardiovascular responses elicited by ~,-aminobutyricacid in the nucleus tractus solitarius: evidence for action at the GABAB receptor, Neuropharmacology, 28 (1989) 515-520. 24 Talman, W.T., Granata, A.R. and Reis, D.J., Glutamatergie mechanisms in the nucleus tractus solitarius in blood pressure control, Fed. Proc., 43 (1984) 39-44. 25 Van der Heyden, J.A.M. and Korf, J., Regional levels of GABA in the brain: rapid semiautomated assay and prevention of postmortem increase by 3-mercapto-proprionic acid, J. Neurochem., 31 (1978) 197-203.