Neuroscience Letters, 100 (1989) 169-174 169 Elsevier Scientific Publishers Ireland Ltd.

NSL 06077

Physiological evidence for l-adrenergic facilitatory control of the cold-induced TRH release in the rat, obtained by push-pull cannulation of the median

eminence

Sandor Arancibia, Lucia Tapia-Arancibia, Helene Astier and Ivan Assenmacher

Laboratory qf Endocrinological Neurobiology, UA 1197 CNRS, University q[" Montpellier-2, Montpellier, (France)

(Received 11 November 1988; Revised version received 29 December 1988; Accepted 2 February 1989)

Key word~." Thyrotropin-releasing hormone; Adrenergic control; Push-pull cannula

The ~-adrenergic antagonists phentolamine and prazosin were administered to male rats to explore their effects on cold-induced TRH release, measured by a chronic push-pull cannula stereotaxically implanted in the median eminence (ME). Phentolamine was given either i.p. (24 or 40 mg/kg), or locally (10 -5 M) in the ME, whereas prazosin was only applied locally (10 -5 M). Phentolamine significantly decreased the cold response (5 + 1 pg/15 min vs 21 __+ 5 pg/15 min; P < 0.02), whatever the administration mode. More- over, the blocking effect of prazosin directly perfused into the ME (11 + 3 pg/15 min vs 26 + 9 pg/15 min; P < 0.05), indicates the specific involvement of ~,-adrenergic receptors in the cold-induced TRH response, and points to the ME as a possible site of facilitatory adrenergic control.

It is well es tabl ished that cold exposure induces an increase o f p l a sma TSH in bo th

rat [8, 15] and h u m a n [13] and tha t ca techolamines are involved in this regulat ion.

However , whereas most studies have shown tha t adrenergic an tagonis t s inhibi t the

co ld - induced TSH increase [9, 14, 18], the effect o f adrenergic agonis ts are more con-

t rovers ia l [10, 12, 17]. This confus ion may well be due to the fact that the measure-

ment o f p l a sma TSH does not d i scr imina te between the different hypo tha l a mic and

p i tu i ta ry sites o f adrenergic regulat ions. The involvement o f T R H med ia t ion in cold-

induced TSH secret ion has been clearly demons t r a t e d [8, 15] and we have measured

a peak o f T R H release in the median eminence (ME) at the beginning o f cold expo-

sure in freely moving unanes the t ized ra ts [1, 2]. In add i t ion , there is morpho log ica l

evidence tha t the pe r ika rya o f the TRH-sec re t ing neurons in the pa raven t r i cu la r nuc-

lei receive noradrene rg ic a n d / o r adrenergic innerva t ion [1 1], while the close p rox imi ty

Correspondence: S. Arancibia, Laboratory of Endocrinological Neurobiology, UA 1197 CNRS, Univer- sity of Montpellier-2, Place E.-Bataillon, 34060 Montpellier, France.

0304-3940,,'89/'$ 03.50 y) 1989 Elsevier Scientific Publishers Ireland Ltd.

170

of both T R H [I 1] and noradrenergic/adrenergic nerve endings [4] in the ME suggests a possible noradrenergic regulation at this distal level. Having recently shown that ~]-adrenergic receptors located in the ME may participate in the control of TRH re- lease [16], we carried out the present investigation to determine whether these .71-ad-

renergic receptors are involved in the cold-induced TRH response, as observed by the push-pull perfusion technique [3].

A stainless-steel cannula was stereotaxically implanted in the ME of 10 male Wis- tar rats (230 -260 g) as previously described [1,2]. Push-pull perfusion was performed one week later according to the standard procedure [2]. Samples of 270/tl were col- lected on ice from tile outer cannula every 15 rain over a total period of 210 min, frozen and stored at - 4 0 ' C until assayed for IR-TRH. Since we know from earlier studies that rats bearing a chronic push-pull cannula in the ME display no alteration in the pattern of the cold-induced T R H surge when explored several times at intervals of a few days [2], in the present study each animal underwent at 4-day intervals, one control sequence with cold exposure alone, and one with cold exposure under drug treatment, in either order. In all animals a preliminary perfusion period of I h with physiological medium alone was monitored to test basal release of TRH. The rats were then quickly transferred from the control room (24 + I 'C) to the cold room at

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4+1 C.

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Fig. 1. Ind iv idual pa t te rns of T R H release in the push-pul l cannu la ted med ian eminence of rats acutely

exposed to cold (4 'C) wi thou t d rugs (control test) or with phen to l amine (Phent. 40 mg /kg i.p.). In (a),

cold exposure a lone preceded the cold + d rug measurements , whereas in (b), the cold + d rug test pre-

ceded the cold test alone.

171

In all drug series, ~-adrenergic blockers were perfused from 45 to 60 min before

and throughout the ensuing period of cold exposure• After histological examination, rats with inadequate cannula locations, or apparent damage in the ventricle were excluded from the study• Phentolamine (Ciba-Geigy) was administered either i.p. (24 or 40 mg/kg) (n = 4), or locally via the push-pull system (i.ME; 10 -5 M; n = 3). Prazo- sin (Pfizer) was administered only locally in the ME (10 -5 M; n = 3). In the i.p. phen- tolamine group, 2 rats were exposed to one cold sequence, and 4 days later, one cold + drug sequence, whereas in 2 other specimens the order of perfusion sequences was reversed• The ME-infused phentolamine and prazosin rats all underwent the stan- dard sequence protocol, while in the latter group, 2 rats underwent a third perfusion sequence with cold alone, 4 days after the cold + drug sequence•

IR-TRH was measured as previously described [16]. Assay sensitivity was 2 pg/200 /~1 of perfusate, and the intra- and interassay coefficients of variation were 2.5 and 4.6 %, respectively• Samples in which IR-TRH was undetectable were assigned the as- say sensitivity value. In the text each mean value relates to total number of measure- ments (n) whereas the n of animals are indicated for each experimental group. Blank assays showed that drugs did not interfer with the assay binding curves. Variance analysis (F-test) was used for statistical comparisons of data.

TRH production rate in control rats maintained at 24°C rarely exceeded the RIA

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Fig. 2. Individual patterns of TRH release in the push-pull cannulated median eminence of rats succes- sively exposed to cold alone (4°C) (a and b, left panels) and to cold after local injection of either phentola- mine (Phent.) (a) right panel or prazosin (Praz.) (b) right panel (10 5 M).

172

sensitivity threshold of 2 3 pg/15 min (Fig. l). On the other hand, in all rats trans-

ferred to the cold room (4:~C) TRH rose sharply within 40.+ 15 min, reaching maxi-

mum values up to 40 pg. A second and weaker rise of TRH was sometimes observed

around 85 min after cold exposure.

Whether administered i.p. (24 or 40 mg/kg; n = 4 ) or locally i.ME through the

push-pull cannula (10 5 M; n = 3), phentolamine per se induced no significant changc

in basal IR-TRH release in the ME (3.9_+0.2, n = 6 8 vs 3.7_+0.1, ;l=51). However,

after transfer to 4 C , the TRH peak observed under standard control conditions was

abolished by i.p. pretreatment with phentolamine (6-+2 pg/15 min vs 22-+6 pg/15

rain in controls: Fig. I). The same effect was obtained when phentolamine (10 5 M)

was given locally (Fig. 2a). Taken together, the mean TRH values after cold + phen-

tolamine in the 7 rats of both groups was 5+ 1 pg/15 min vs 21 +5 pg/15 rain after

cold alone ( P< 0.02) (Fig. 3). Local perfusion of the ej-adrenergic antagonist prazosin, (10-5 M; n = 3; Fig. 2b)

at 2 4 C provoked no change in basal IR-TRH release in the ME, but the treatment

dramatically reduced or abolished the cold-induced TRH peak (l l .+ 3 pg/l 5 rain vs

26.+9 pg/15 min; P < 0.05). Our data first show that push-pull cannulation of the ME is well suited to direct

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TRH pgllsmin.

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o l o l o4 o l o4 o l e l oq o l o l o l o l o l

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C T C T Phent. Praz.

Fig. 3. TRH release (mean + S.E.M.) in the push-pull cannulated median eminence of rats exposed to cold alone (C) or to cold under phentolamine (Phent., i.p. or local) or prazosin (Praz., local) treatment (T), White bars indicate basal TRH releases, and the filled bars indicate additional peak TRH releases after cold exposure; number of animals in parentheses.

173

evaluation of the effects of systemically or locally administered adrenergic antago- nists on cold-induced TRH release. It is unlikely that a significant diffusion of drugs occurs, which would affect other neuroendocrine structures involved in TRH secre- tion. Regarding the i.p. route used for phentolamine, a predominantly peripheral blocker [9], the short time span of the experiment may avoid major penetration of the drug outside the ME, and a significant diffusion of physiologically active concen- trations would presumably require higher doses and a lower turnover than our flow

rate. The blocking effect of i.p. phentolamine and of i.M.E phentolamine and prazosin

strongly favors a physiological role for the ~:catecholamine binding sites located on the TRH nerve endings in the ME [16], according to the concept that neurotransmit- ter-containing nerve endings may release their chemical signals into the adjacent extracellular fluid and affect neighboring axons [5]. Cytochemical evidence of a rich catecholaminergic innervation of the ME [4] and the occurrence of alpha-adrenergic receptors in the stalk ME [6] also argue in favor of a noradrenergic action at this level.

So far the role of adrenergic signals in the thyrotropic axis essentially deals with TSH rather than TRH measurements. This more peripheral site of exploration which may include a regulation at the pituitary level [7] together with consistent differences in dosages and administration routes of the drugs may explain the discrepancies in the literature. Whereas systemic or i.c.v, administered phentolamine or prazosin were claimed to be ineffective on basal and cold-induced TSH release [9, 10, 14], phentola- mine [18] and phenoxybenzamine [9, 12, 18] as well as ~l- [17], and c~2-blockers [10, 17] markedly depressed cold-induced TSH secretion, which fits well with our present findings on TRH release. Furthermore, the basal secretion of TSH was found to be decreased by i.c.v, administered noradrenaline [12], or the ~:agonists methoxamine and phenylephrine [10] and by the 0~2-agonist cionidine [12], whereas the latter drug is said to enhance baseline TSH release [10, 17].

To summarize, our present study clearly indicates a facilitatory role of the ~l-adre- nergic receptors located in the ME in the cold-induced TRH release. Clearly how- ever, other and possibly opposite catecholaminergic regulations may occur at other sites of the system.

This work was supported in part by the INSERM, Grant 884002.

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