Displacement of norepinephrine by α-methylnorepinephrine in the nucleus tractus solitarius of the rat

ELSEVIER

Neuroscience Letters 184 (1995) 59-62

Displacement of norepinephrine by cr-methylnorepinephrine in the nucleus tractus solitarius of the rat Gordon F. Andersona,*, Carolyn Clough-Helfmanb,

Robin A. Barracob

aDepartment of Pharmacology, Wayne State University, School of Medicine, Detroit, MI 48201, USA bDepartment of Physiology, Wayne State University, School of Medicine, 540 E. Canfield, Detroit, MI 48201, USA Received 6 September 1994; revised version received 13 November 1994; accepted 15 November 1994

Abstract

Rat brain slices from. the dorsomedial medulla containing the nucleus tractus solitarius were loaded with [3H]norepinephrine ([3H]NE) for superfusion. Electrical stimulation (3 Hz, 25 mA, 1 min) resulted in fractional release ratios S&S, of 0.97 f 0.02 in normal Krebs-Henseleit (KH) and 0.93 f 0.06 in the presence of 30pM cocaine. With cocaine in the KH medium, L-CC methylnorepinephrine (a-MeNE) significantly reduced the [3H]NE release S2/S1 without affecting the basal release ratios. Without cocaine in the KH medium both 0.1 and l.OpM a-MeNE increased the basal release B2/B1 that was not affected by yohimbine. Prazosin had no effect on the S2/S1 ratio but did attenuate the basal release effects of a-MeNE. In low Ca2+ studies where the S2 stimulus was abolished, 1.OpM a-MeNE induced a sharply elevated increase in the B$Bt ratio. It appears that a-MeNE in the presence of the uptake inhibitors reduces presynaptic neurotransmitter release through az-adrenoceptors, whereas when uptake of the monoamines was not blocked a-MeNE was considerably more efficacious as a displacing agent of neurotransmitter. Keywords:

cr-Methylnorepinephrine; Transporter; Norepinephrine release; Nucleus tractus solitarius; Cardiovascular control

The nucleus of the tractus solitarius (NTS) serves primarily as a sensory relay center for visceral, respiratory and cardiovascular afferent neural pathways. Most of the common neurotransmitters and neuromodulators identified in various areas of the central nervous system, have also been described within the NTS, as well as the respective receptors for each of the transmitter substances, and has been recently reviewed [8]. Nbrepinephrine has been shown to prdduce prominent physiological responses when micro-injected into various levels of the NTS especially in the control of cardiovascular function. Micro-injections of incremental doses of l-5 nmol of NE! into the NTS produced a dose dependent decrease in blood pressure and heart rate that was blocked by phentolamine [2], while larger concentrations of NE! produce pressor responses thclught to be mediated through aladrenoceptors [4,5]. ‘Various receptor blocking experiments for specific adrenoceptors have been employed in an effort to describe the receptor subtype associated with the heart rate and blood pressure changes. Micro*Corresponding

author, Tel: +l 313 5771537; Fax: +l 313 5776739.

injections of prazosin produced no response, and yohimbine, the a2-adrenoceptor antagonist, promoted an increase in blood pressure suggesting that the increased NE release associated with yohimbine blockade of presynaptic receptors was acting postsynaptically at aIadrenoceptors sites [‘7]. When a-methylnorepinephrine (a-MeNE) was micro-injected into the NT.& a decrease in blood pressure was produced, which would be consistent with an a2-adrenoceptor agonist, a response that would reduce synaptic levels of NE [3]. It was the purpose of this study to examine the effects of a-MeNE on the presynaptic contrql and release of NE using [3H]norepinephrine ([3H]NE) labeled brain slices from the NTS. a-MeNE has been consistently described as an a2-adrenoceptor agonist producing autoinhibition of NE! release during nerve stimulation. Male Charles River rats between 300 and 350 g were immobilized with light diethyl ether anesthesia and decapitated. The brain was rapidly removed and transferred to 4°C modified Krebs-Henseleit solution (KH), in mM: 117 NaCl; 4.6 KCl; 10.0 glucose; 25.0 Na2HC03; 1.2 KH,PO,; 2.0 CaCl,; and 1.2 MgS04 (pH 7.4) aerated

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G.F. Anderson et al. / Neuroscience Letters 184 (1995) 59-62

with 95% oxygen and 5% carbon dioxide. The brain stem was dissected free from the surrounding tissue under chilled KH, and the medullary portion was then isolated by transecting the brain stem and spinal cord. The dorsomedial portion of the medulla containing the nucleus tractus solitarius (NTS) was bisected coronally from the ventral portion of the brain stem and further blocked with sagittal transections along both sides of the dorsal medulla. The freshly dissected tissue was transferred to an ice-chilled plate for sectioning, and the area postrema was gently removed. Consecutive slices 400/gin thick were prepared with a Mcllwain tissue slicer cut transversely from the dorsomedial portion of the medulla beginning at a level just caudal to the posterior tip of the calamus scriptorius, and continuing cephalically to the obex [1]. These slices contained portions of the vagal motor nucleus and twelfth cranial nucleus in addition to the NTS. Slices were warmed to 37°C and equilibrated for 30 min in aerated KH buffer. Slices were then incubated with 0.1/zM [3H]norepinephrine, [3H]NE, 13.8 Ci/mmol for 30 min at 37°C in a water bath. Following the isotope loading, the slices were rinsed twice for 30 min in 150 ml of 37°C KH solution. Single NTS tissue slices were transferred to each superperfusion chamber, Brandel SF 12, having two platinum stimulating grids 1 cm apart. Each perfusion chamber had a 0.3 ml volume, and was maintained at 37°C. The baths were superperfused continuously at 0.5 ml/min with aerated KH. Tissues loaded with [3H]NE were perfused with medium containing either 30.0/zM cocaine to block the re-uptake of NE or in KH without cocaine as described in the results. Perfusate samples were collected at 5-min intervals beginning 55 min after the superperfusion was started. Slices were stimulated for 1 min at two time intervals 55 min apart, S 1 (75 min) and S 2 (130 min), and all drug additions were started at 110 min into the perfusion except where described differently in the results. The stimulus pulses were square wave 25 mA, 2 ms duration at 3 Hz, for 1 min generated by a Brandel ES-112 stimulator. In low calcium experiments, the CaC12 was lowered to 0.03 mM in the KH perfusion medium and changes in the buffer were made at 100 min into the perfusion after all of the Sl stimulus samples had been collected. The perfusate fractions were counted by liquid scintillation (LS) in a Beckman LS3801 in Safety Sol, RPI, for 5 min. The tissues were removed from each perfusion bath at the end of the experiments and solubilized in 1 ml of Solvable for 5 min prior to adding the Safety-Sol LS medium. Results are reported as the fractional release of the total labeled amount of [3H]NE remaining for each of the collection intervals. The evoked stimulated release was determined by the sum of the fractional release for the 15min period after the stimulus train minus the corrected background release. The background release was corrected at each collection interval by using the slope of the sample immediately prior to stimulation and the sample

collected 20 min after the stimulus. Data are reported as the ratios of the total stimulated fractional release as $2/S 1, and background release as the ratio of B2/B 1 from the 5-min sample collected just prior to each of the stimuli. The significance (P < 0.05 or less) was determined by unpaired Student's t-test, A N O V A and Scheffe analysis was used for post hoc comparisons between experimental and control groups. Drugs for this study; tyramine, yohimbine, L-a-methyl norepinephrine and clonidine were obtained from RBI Research Biochemicals Inc., Natick, MA; and [3H]norepinephrine and Solvable, Dupont NEN, Boston M A 02118, USA. Experiments began 55 min after the start of the superperfusion and the mean tissue loading for all control slices was 68 372 dprri per slice at this fraction collection. The stimulated releasa, $2/$1 ratio, for [3H]NE, without cocaine in the KH medium was 0.97 _+0.02, and the total fractional release above background following the stimulation was 1.43 _+0.09 in control groups, with 17.4 _+ 0.5% of the label released during the course of the experiment. When 30/zM cocaine was included in the KH the $2/S l ratio was 0.93 _+0.06 and the total fractional release for the 1 min stimulation train was increased to 2.05 _+0.15, a 43% increase in the evoked release (Table 1 and Fig. 1A) with 23.0 _+2.1% of the labeled ligand release during the course of the experiments. Basal Table 1 [3H]Norepinephrine release

from

Control Control with cocaine Yohimbine 0.1/aM with cocaine Clonidine 0.1/aM with cocaine a-MeNE -0.1/aM - 0.1/aM with cocaine - 0.1/~M and yohimbine 0.1/aM - 0.1/aM and prazosin 0.01/aM - 1.0/aM - 1.0/aM with cocaine -1.0/aMandyohimbine0.1/aM - 1.0/aM and prazosin 0.01/aM Tyramine - 10/aM - 10/aM with cocaine Low Ca2+ 0.03 mM a-MeNE 1.0/aM/low Ca2+

the NTS $2/SI ratio

B2/B1 ratio

N

0.97 + 0.02 0.93 + 0.06 1.87+ 0.02** 0.73+ 0.03**

0.88 + 0.02 0.83 + 0.05 0.88 ± 0.03 0.87 + 0.03

12 5 4 10

0.69+0.04** 0.70 + 0.05** 1.44+0.03"* 0.72_+0.04* 0.48_+0.12"* 0.37 ± 0.02** 1.35±0.13"* 0.43± 0.08**

1.16+0.04"* 0.86 + 0.02 1.12+0.04"* 0.96 + 0.02** 4.39 ± 0.98** 1.14 ± 0.13 3.02+0.53** 2.11 ± 0.20**

8 8 5 6 5 6 5 4

1.01 ± 0.03 1.06 ± 0.03 0.02±0.01"* 0.07 ± 0.04**

5.24 :e 0.03* 2.14 + 0.28* 0.96±0.11 6.10 ± 1.36"*

2 3 4 4

Summary o f the stimulated and basal release o f [3H]norepinephrine from rat brain slices, containing primarily nucleus tractus solitarius. Both S 1 and S 2 stimuli were 3 Hz, 25 mA, 2 ms for 1 rain. Basal release, B2/B 1 ratio, is described in the text. a-Methylnorepinephrine, a MeNE, at reported concentrations. Experiments with cocaine contained 30/aM cocaine in KH medium, present for the entire duration of the perfusion experiment. Low Ca2+ experiments were performed in KH with 0.03 mM calcium. Results are reported as the mean + SEM. * P < 0.05 and ** P < .01 from controls unpaired Student's t-test or

ANOVAanalysis.

G.F. Anderson et al. I Neuroscience Letters 184 (1995) 59--62

8

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Fig. 1. Percent fractional release of [3H]norepinephfine, [3H]NE, from bITS slices in single representative experiments. (A,B) From tissues with 30/tM cocaine present in KH; (C,D) without cocaine present. (D) The extracellular peffusion medium level of Ca2+ was lowered to 0.03 mM at 100 min.

release of [3H]N~, B2/B 1, was between 0.88±0.02 for normal KH perfusion medium and 0.83 _+0.05 with cocaine containing KH medium in control tissues (Table 1). Initial experiments were performed with clonidine and yohimbine with cocaine present in the KH medium. When 0.1 # M clonidine was introduced into the perfusion the $2/$1, ratio was reduced 25% (P<0.01) for the evoked release of [3H]NE, without a significant alteration in the B2/B1 ratio from control slices. Yohimbine, 0.1 #M, sharply increased the $21S1 ratio by 201% of the control (P < 0.01) and the B2/B l ratio was not significantly affected (Table 1). Low calcium control studies, not shown, were performed with incremental lower calcium concentrations, ranging from 2.0 mM down to 0.01 mM in the KH medium to find an appropriate low calcium concentration that would abolish the S2 exocytotic release (Table 1 and Fig. 1D). In these experiments the calcium was lowered in the perfusic,n medium at 100 min, 20 min after $1, and maintained at that concentration for the duration of the experiment. The $2 stimulus train was moved to 155 min to allow for extracellular equilibration of the lower calcium concentration. It was clear from the low calcium response stud)' that the evoked transmitter release was calcium dependent, and that the $2 response was virtually abolished at or below calcium concentrations below

61

0.03 mM within 55 min of exposure, and the B2/B 1 ratio was unaffected (Table 1 and Fig. I). When either 0.1 or 1.0/zM a-MeNE was introduced into the KH medium 110 min into the superfusion in the presence of 30/zM cocaine, the $2/S1 ratio was reduced to 76% at 0.1/zM and to 40% at 1.0/zM of the control responses (P > 0.01) suggesting aE-adrenoceptor presynaptic inhibition, (Table 1 and Fig. 1). The B2/B ! ratio was not effected at 0.1/~M and slightly elevated to 1.14, but was not significantly different from control tissues. When the cocaine was omitted from the perfusion medium the addition of either 0.1 or 1.0#M a-MeNE promoted a significant increase in the basal release of [3H]NE within the first fractional sample and continued to increase the basal release for the remaining perfusion period. The B2/B 1 ratio in these studies was increased 32% for 0.1/zM and 353% for 1.0/zM (P < 0.01) over the control release ratio, and resulted in a 33.3 ±2.5% total release, at 1.0/zM, of the [3H]NE from the slices during the course of the experiment (Fig. 1C). With 0.1/zM yohimbine added to KH medium together with either 0.1 or 1.0/~M a-MeNE the $2/S 1 ratio inhibition was blocked while the increased basal release was unaffected, and 0.01/zM prazosin had no effect on either $2/S 1 or B2/B1 ratios (Table 1). Similar experiments with tyramine over a range of 0.1-100/zM (not completely shown in Table 1) exhibited the same actions, when 10.0/zM tyramine was added to the perfusion medium it produced approximately the same magnitude increase in basal release and had considerably lesser effects at 1.0/~M compared to the 1.0/zM aMeNE (Table 1). Although tyramine promoted the displacement of [3H]NE from the slices it did not reduce the $2/S I ratio, and therefore did not demonstrate significant a 2 adrenoceptor agonist activity at the concentrations employed. The effects of 1.0/zM tyramine on basal release were also abolished when cocaine was included in the KH medium (Table 1). When the normal perfusion medium was substituted with 0.03 mM calcium KH, after $1 and 1.0/zM a-MeNE was introduced into the perfusion medium at 140 min, the basal release of [3H]NE increased within the first 5 min sample, and continued rising as in the earlier experiments with normal calcium concentrations. The B2/B i ratio under the low calcium condition was increased to 6.10 (P<0.01) and the $2 stimulus release of [3H]NE was nearly abolished, $2/$I 0.07 (P < 0.05) (Table 1 and Fig. 1D). The present study provides evidence for a-MeNE promoting both auto-inhibition of stimulated release of NE through (a2-adrenoceptors that was blocked by yohimbine, and the displacement of stored NE through the presynaptic neuronal transporter in [3H]NE loaded NTS brain slices. The evoked release of NE was calcium dependent, while the drug-induced basal release of neurotransmitter by the respective agonists was not calcium dependent. Further, the basal release changes mediated by

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G.F. Anderson et al. I Neuroscience Letters 184 (1995) 59-62

( a - M e N E were blocked by the presynaptic transport inhibitor cocaine. Tyramine in our studies had a tenfold lower affinity than ( a - M e N E for its displacing action of [3H]NE. That these actions were similar was further supported by the observation that both tyramine and a - M e N E induced release were blocked by cocaine, and both increased the basal efflux o f neurotransmitters in low calcium environments, even when the $2 stimulated exocytotic release was nearly abolished. It is conceivable that agents like a - M e N E that reduce blood pressure when micro-injected or are administered chronically do so by dual mechanisms involving both a 2adrenoceptor inhibition of NE release, and a secondary longer term effect may be related to the presynaptic transporter and displacement o f NE from vesicle storage sites within the neuron promoting a gradual depletion of neurotransmitter. This work was supported by: N I M H (MH 47181 A N D M H 17153), N I H (GM-08167). N S F (HRD 9104797).

[1] Barraco, R., EI-Ridi, M.R., Ergene, E., Parizon M. and Bradley D., An atlas of the rat subpostremal nucleus tractus solitarius, Brain Res. Bull., 29 (1991) 703-765. [2] De Jong, W., Noradrenaline central inhibitory control of blood pressure and heart rate, Eur. J. Pharmacol., 29 (1974) 179-181. [3] De Jong, W. and Nijkamp, F.P., Centrally induced hypotension and bradycardia after administration of alphamethylnoradrenaline into the area of the nucleus tractus solitadi of the rat, Br. J. Pharmaeoi., 58 (1976) 593-598. [4] Kubo, T. and Misu, Y., Pharmacological characterization of the a-adrenoceptors responsible for a decrease of blood pressure in the nucleus tractus solitarii of the rat, Nannyn-Schmiedeberg's Arch. Pharmacol., 317 (1981) 120-125. [5] Philippu, A., Regulation of blood pressure by central neurotransmitters and neuropeptides, Rev. Physiol. Bioehem. Pharmacol., 111 (1988) 1-15. [6] Pratt, G.D. and Bowery, N.G., The 5-HT3 receptor ligand, [3H]BRL 43694, binds to presynaptic sites in the nucleus tractus solitarius of the rat, Neuropharmacology, 28 (1989) 1367-1376. [7] Tung, C.S., Goldberg, M.R., Hollister, A.S. and Robertson, D., Both a- and fl-adrenoceptors contribute to the central depressor effect of catecholamines, Brain Res., 456 1988) 64--70. [8] Van Giersberzen, P.L.M., Plakovits, M. and De Song, W., Involvement of neurotransmitters in the nucleus tractus solitarii in cardiovascular regulation, Physiol. Rev., 72 (1992) 739-824.