Regulatory Peptides, 37 (1992) 167-180 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0167-0115/92/$05.00

167

REGPEP 01134

Participation of pertussis toxin-sensitive GTP-binding regulatory proteins in the suppression of baroreceptor reflex by neurotensin in the rat M.J. Fu ~, K.S. Lin l, Julie Y . H . Chan 2 and Samuel H . H . Chan 1 IInstitute of Pharmacology, National Yang-Ming Medical College, Taipei (Taiwan, R.O.C.) and 2Department of Medical Research, Veterans General Hospital-Taipei, Taipei (Taiwan, R.O.C.) (Received 19 July 1991; revised version received 15 October 1991; accepted 16 October 1991)

Key words: Neurotensin; B aroreceptor reflex; Guanine nucleotide-binding regulatory protein; Gi; Gp; Cholera toxin; Forskolin; Pertussis toxin; N-Ethylmaleimide; Phorbol ester; Rat

Summary We evaluated the molecular mechanism that may underlie the suppressive effect of neurotensin (NT) on the baroreceptor reflex (BRR), using Sprague-Dawley rats that were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). Intracerebroventricular (i.c.v.) application of NT (15 nmol) significantly inhibited the BRR response. Such an inhibition was appreciably antagonized by pretreating animals with i.c.v, injection of pertussis toxin (10 or 20 pmol), N-ethylmaleimide (1 or 2 nmol), forskolin (30 or 60 nmol) or phorbol 12-myristate 13-acetate (2 or 4 nmol), but not by cholera toxin (15 or 30 pmol). More specifically, pretreatments with bilateral microinjection into the nucleus tractus solitarius (NTS) of pertussis toxin (80 or 160 fmol), N-ethylmaleimide (80 pmol), forskolin (480 pmol) or phorbol 12-myristate 13-acetate (16 or 32 pmol) also blunted the NT-induced suppression of BRR, although cholera toxin (120 or 240 fmol), or 1,9-dideoxyforskolin (480 pmol) had no appreciable effect. These results suggest that a pertussis toxin-sensitive guanine nucleotide-binding regulatory protein(s), which is not likely to be Gs, possibly Gi or Gp, may be involved in the transmembrane signaling process that underlies the suppression of BRR response by NT at the NTS.

Correspondence: S.H.H. Chan, Institute of Pharmacology, National Yang-Ming Medical College, Taipei 11221, Taiwan, Republic of China.

168 Introduction

Neurotensin (NT) is a tridecapeptide that was originally isolated from bovine hypothalamus by Carraway and Leeman [1] in 1973 and has over the years been found to be involved in many physiologic processes [2-6]. In a recent study [7], our laboratory demonstrated that the endogenous NT in the brain may exert a tonic inhibitory action on the baroreceptor reflex (BRR) response, the most fundamental mechanism in central cardiovascular regulation [8]. Furthermore, we identified that one of the brain sites that may be involved in this modulatory action of NT is the nucleus tractus solitarius (NTS), the terminal site of baroreceptor afferents [9,10]. An interesting observation in our study [7] was that whereas NT has a rather short half-life [ 11 ], its suppressive effect on the BRR response endured for more than 20 min. One possible explanation is that this particular action of the tridecapeptide may involve the participation of second messengers. The involvement of both cAMP and inositol phospholipid pathways in the molecular mechanism of NT has already been reported in other test systems. For example, intracerebroventricular application of NT decreases cAMP and increases c G M P levels in the hypothalamus [ 12]. The tridecapeptide also stimulates the hydrolysis of inositol phospholipids in brain slices [ 13 ]. In neuroblastoma N 1 E l l 5 cells [14-16], NT elicits a decrease in cAMP and an increase in inositol triphosphate and c G M P levels, in a process that may involve a pertussis toxin-sensitive guanine nucleotide-binding regulatory protein. It is now rather firmly established that subtypes of guanine nucleotide (GTP)-binding regulatory proteins exist [17,18] in the control of diverse pathways of transmembrane signaling. Also available are several pharmacologic tools for the evaluation of the participation of these regulatory proteins or their associated second messenger systems in biologic processes. For example, cholera toxin prolongs the action of Gs by ADPribosylation of its e-subunit and suppression of GTPase activity [ 19,20]. Since forskolin increases the cAMP level by directly activating adenylate cyclase [21,22], it is often used to confirm the participation of Gs in a biologic process. Recent reports [23,24], however, suggest that forskolin may also directly activate the voltage-dependent K + channels. 1,9-Dideoxyforskolin, an analog of forskolin that directly stimulates voltagedependent K + channels without involving adenylate cyclase, can be used to identify this mode of action [24]. Pertussis toxin, which is frequently used to inactivate Gi [25,26] by ADP-ribosylation of its :~-subunit, is now also known to inactivate Gp and Go by the same mechanism [27-29]. Two agents can be used to further differentiate the involvement of these three subtypes of GTP-binding regulatory proteins in biologic processes. N-Ethylmaleimide, which at low doses uncouples Gi from G T P by alkylation [30-32] and phorbol ester, which directly stimulates protein kinase C and causes a feedback inhibition on polyphosphoinositides or Gp [33-35]. The present study was undertaken, using the above pharmacologic tools, to evaluate the molecular mechanisms that may underlie the suppressive action of N T on the BRR response. We conclude that a pertussis toxin-sensitive GTP-binding regulatory protein(s), which is not likely to be Gs, possibly Gi or Gp, may be involved in the signal transduction process that underlies the modulation of the BRR response by NT at the NTS.

169 Materials and Methods

General preparation Adult, male Sprague-Dawley rats (219-260 g), anesthetized with sodium pentobarbital (50 mg/kg, i.p., with 10 mg/kg/h i.v. supplements), were used in this study. The trachea in each animal was intubated to facilitate ventilation, and the right femoral artery and vein were cannulated for the measurement of systemic arterial pressure and introduction of drugs. Pulsatile and mean systemic arterial pressure, and heart rate monitored via a cardiotachometer triggered by the arterial pulses, were routinely displayed on a Grass polygraph.

Intracerebroventricular (i.c. v.) injection Following the procedures used in previous studies [7,36-39], a 25-gauge stainlesssteel cannula was implanted into the lateral cerebral ventricle on the right side at (mm): P0.8-1.0, R1.4-1.7 and H3.5-4.5 with reference to the bregma. I.c.v. injection of NT, cholera toxin, forskolin, pertussis toxin, N-ethylmaleimide, phorbol 12-myristate 13-acetate, artificial cerebrospinal fluid (aCSF, as vehicle) or 75 ~o ethanol (vehicle for forskolin) was carried out by inserting a 27-gauge, flatly bevelled needle, into the guide cannula. The former was connected to a 10 #1 Hamilton microliter syringe by a PE-20 polyethylene tubing. A total volume of 5/~1 was delivered over at least 1 min to allow for full diffusion of the solution.

Microinjection of chemicals Direct microinjection of cholera toxin, forskolin, 1,9-dideoxyforskolin, pertussis toxin, N-ethylmaleimide or phorbol 12-myristate 13-acetate into the NTS was carried out, using a micropressure infusion pump (Narashigi) and through a glass micropipette. The stereotaxic coordinates for the NTS were (mm): - 0.80 to + 0.50 from, and 0.35 to 0.50 lateral to the obex; 0.35 to 0.80 below the surface of the medulla. A total volume of 20 nl was delivered into each side of the NTS. Direct injection of aCSF or 75~o ethanol, at the same volume, served as the vehicle control.

Evaluation of baroreceptor reflex response The sensitivity of baroreceptor reflex (BRR) response was evaluated by the ratio method [ 7,36,38,39 ]. Arterial baroreceptors were stimulated by a transient hypertension induced by an intravenous injection of phenylephrine (5/~g/kg). The quotient that represents unit reflex decrease in heart rate per unit increase in mean systemic arterial pressure (b/min per mmHg) was used as the index for the BRR response. The temporal effect of different chemical treatments on the BRR response was evaluated at 10-min intervals during the 60-65 min postinjection. The quotient at each time point was normalized to a percentage of pretreatment control to compensate for variations between animals, and allow for comparison between different treatment groups.

Experimental procedures The body temperature of animals was maintained at 37 °C throughout the experiment with a thermostatically-regulated heating pad. Rats were also artificially ventilated to avoid possible confounding artifacts arising from respiratory disturbance.

170 Our first series of experiments investigated the action of i.c.v, administered cholera toxin (15 or 30 pmol), forskolin (30 or 60 nmol), pertussis toxin (10 or 20 pmol), N-ethylmaleimide (1 or 2 nmol) or phorbol 12-myristate 13-acetate (2 or 4 nmol) on the effect of NT (15 nmol, i.c.v.) on the sensitivity of BRR response. In these experiments, BRR was induced before and 60 min after each pretreatment, and during the first 5 rain of every 10-min interval over 60-65 min following i.c.v, administration of NT. These same procedures were repeated in our second series of experiments, except that cholera toxin (120 or 240 fmol), cholera toxin (240 fmol) plus 0.1 ~o Triton X-100, forskolin (240 or 480 pmol), 1,9-dideoxyforskolin (480 pmol), pertussis toxin (80 or 160 fmol), N-ethylmaleimide (16 or 80 pmol), or phorbol 12-myristate 13-acetate (16 or 32 pmol) was microinjected bilaterally into the NTS. Chemica& All chemicals used in the present study were obtained from Sigma (St. Louis, U.S.A.). Solutions of NT, cholera toxin, forskolin, 1,9-dideoxyforskolin, pertussis toxin, N-ethylmaleimide and phorbol 12-myristate 13-acetate were thawed, and phenylephrine freshly prepared, immediately before the experiment, aCSF served as the vehicle for all chemicals, except with forskolin and 1,9-dideoxyforskolin, when 75~o ethanol was used. Histology The brain was removed after each experiment and fixed in 30 ~o sucrose-10 ~o formaldehyde-saline solution for at least 48 h. Histologic verifications of the position of i.c.v. injection or microinjection sites were carried out on frozen 25-/zm sections stained with Cresyl violet. Identification of microinjection sites in the NTS was aided by the addition of 1 ~o Evans blue in the injection medium. Stat&tics The effects of various treatments on the sensitivity of BRR response were statistically assessed using the analysis of variance (ANOVA). This was followed by the StudentNewman-Keuls test for a posteriori comparisons of the means as appropriate. P < 0.05 was taken to indicate statistical significance.

Results Actions of i.c. v. admin&tration of chem&als on the effect of neurotensin on the baroreceptor retie x N T (15 nmol, i.c.v.) elicited a significant inhibition of the BRR response in animals that received i.c.v, pretreatment of either vehicle (Figs. 1-5). As reported in our previous study [7], such a suppressive effect maximized at 5-10 rain postinjection. The BRR response to NT at this postinjection interval was therefore used in our subsequent analysis of the actions of various pretreatments. I.c.v, administration of cholera toxin (15 or 30 pmol) did not by itself produce discernible effect on the BRR (Fig. 1). At the same time, this toxin (15 or 30 pmol) also

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did not elicit appreciable influence on the suppressive effect of NT (15 nmol, i.c.v.) on the BRR response (Fig. 1). Pretreatment with i.c.v, administration of forskolin (30 or 60 nmol) (Fig. 2), pertussis toxin (10 or 20 pmol) (Fig. 3), N-ethylmaleimide (1 or 2 nmol) (Fig. 4) or phorbol 12-myristate 13-acetate (2 or 4 nmol) (Fig. 5) resulted in significant antagonism of, and in most cases, reversal of, the inhibition by NT (15 nmol, i.c.v.) on the BRR response. These respective treatments, on the other hand, did not by themselves appreciably affect the same reflex (Figs. 2-5, Table I).

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Participation of pertussis toxin-sensitive GTP-binding regulatory proteins in the suppression of baroreceptor reflex by neurotensin in the rat.

We evaluated the molecular mechanism that may underlie the suppressive effect of neurotensin (NT) on the baroreceptor reflex (BRR), using Sprague-Dawl...
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