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

120I 80~

200[ 16o[ I

8ok

"l-

300~

tP4 nmol c

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

E E

120[

120 80"

801-

~~

160

2°°f

.T E 160 E 120 ~ ~E 80

200' 160 f

~" 120