ovarian steroid modulation of neurokinin contents in hypothalamus, pituitary, trigeminal nucleus,...

Journal of Neuroendocrinology, 1998, Vol. 10, 823–828

Ovarian Steroid Modulation of Neurokinin Contents in Hypothalamus,Pituitary, Trigeminal Nucleus, and Cervical Spinal Cord of theOvariectomized Female Rat

P. Duval, V. Lenoir and B. KerdelhueLaboratoire de Neurobiologie Cellulaire et Moleculaire, CNRS EP 1591, Faculte des Sciences Pharmaceutiques et Biologiques, Paris, France.

Key words: substance P, neurokinin A, steroids, gonadotrope function, nociception.

Abstract

Ovariectomized rats were treated with estradiol benzoate (EB) and progesterone in conditionsknown to negatively and positively regulate gonadotropin secretion. Injection with EB decreased theplasma concentration of substance P at the time of the positive feed-back exerted by EB ongonadotropin secretion, while having no effect on the plasma concentration of neurokinin A. In thehypothalamus, EB injection enhanced the substance P and neurokinin A content, while progesteronereduced the substance P content. In the anterior pituitary, the substance P content was increasedafter progesterone, and this increase was blocked by EB. Conversely, in the posterior pituitary, thesubstance P content was reduced after progesterone, and this effect was enhanced by EB. In thetrigeminal nucleus, the substance P content was increased after progesterone and EB, while onlyprogesterone affected neurokinin A content. Finally, in the cervical spinal cord, the substance P andneurokinin A contents were reduced after EB. We conclude that neurokinin contents are controlledby ovarian steroids not only in the hypothalamo-pituitary complex but also in the trigeminal nucleusand the cervical spinal cord.

Substance P and neurokinin A are present both in the anterior the whole hypothalamus are low during diestrous II andhigh during the preovulatory LH and FSH surges (14).and posterior pituitary (1, 2) and in the hypothalamus (2),

and are involved in regulating the secretion of pituitary Conversely, in the anterior pituitary, the neurokinin contentis high during diestrous II and at the beginning of proestrous.hormones, including LH and FSH (3–5). In the trigeminal

nucleus and the cervical spinal cord, neurokinins are involved During the estrous cycle in the rat, these variations arepositively and negatively correlated with the plasma concen-in nociception. Substance P enhances the activity of neurones

implicated in nociception (6), and its action is controlled by trations of E2 and progesterone. In ovariectomized (ovx)female rats, some studies have shown that E2 can induce anendogenous opioids (7). Both of these functions are regulated

by ovarian steroids. The actions of estradiol 17b (E2) and increase of the hypothalamic substance P content (15, 16)while another study, in different conditions, showed no effectprogesterone on the hypothalamo-pituitary complex are well

documented, but E2 and progesterone also seem to be (2). At the pituitary, substance P and neurokinin A areup-regulated by E2 (2, 17, 18). Frankfurt et al. (19) haveinvolved in the regulation of nociception (8, 9). Both E2 and

progesterone enhance the pain threshold during pregnancy also demonstrated variation of the substance P content inthe septum and the amygdala. We have shown that trigeminaland parturition via effects on opioid systems (10, 11).

During the rat estrous cycle, the substance P contents of nucleus content of neurokinins does not vary during theestrous cycle, but in the cervical spinal cord there is a decreasethe median eminence and the medial preoptic nucleus are

high on the day of proestrous (12), whereas, in the arcuate in these contents during the first days of the estrous cycle (20).In the present work we measured substance P and neuroki-nucleus, where substance P is synthesized (13), the content

is high during diestrous I. Furthermore, we have recently nin A contents in the hypothalamus, the pituitary, the trigem-inal nucleus and the cervical spinal cord after estradiolshown that the substance P and neurokinin A contents of

Correspondence to: P. Duval, Laboratoire de Neurobiologie Cellulaire et Moleculaire, CNRS EP 159, Faculte des Sciences Pharmaceutiques etBiologiques, avenue de l’Observatoire, 75270 Paris cedex 06, France.

© 1998 Blackwell Science Ltd

824 Neurokinins and ovarian steroids

benzoate (EB) and/or progesterone injection in experimentalconditions which induce both negative and positive feed-backactions on LH secretion, in the ovx female rat.

Results

One group of ovx female rats was injected with sesame oil(the ovarian steroid vehicle) and killed 24 or 33 h later. Asecond group was injected with EB(50 mg/kg) and killed 24or 33 h later. A third group was injected with oil at time 0and with progesterone(6 mg/kg) 24 h later, and were killed33 h after time 0. The last group was injected with EB attime 0 and with progesterone 24 h later, and were killed 33 hafter EB injection

Plasma concentrations

Plasma LH concentrations were lower 24 h after EB injectionthan in oil-injected rats (1.46±0.26 ng/ml vs 3.50±0.35 ng/ml; P<0.05), while after 33 h, they were higher (6.47± Time after EB injection

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0.24 ng/ml vs 3.60±0.26 ng/ml; P<0.05). ProgesteroneF. 1. Hypothalamic GnRH content after vehicle (%), Estradiol benzo-injection had no significant effect. In rats treated withate (b), Progesterone (a), or Estradiol benzoate and Progesterone (q)EB+progesterone, the LH concentration was higher 33 h treatment. Values were considered as different if P>0.005 (*).

after EB injection (13±0.45 ng/ml vs 6.47±0.24 ng/ml forEB-treated and 3.6±0.26 ng/ml for oil-treated rats; P<0.05).

The plasma concentration of substance P was higher 24 hafter injection of EB than in oil-treated rats (6.13±0.21 ng/ml in progesterone-injected rats than in all other groups at thisvs 4.02±0.22 ng/ml; P<0.05). Thirty-three hours after injec- time (Fig. 3). Injection with neither EB nor progesterone hadtion there were no significant differences between any groups. any significant effect on neurokinin A content. However, theNo significant changes were observed in the plasma concen- content in rats treated with progesterone alone was highertration of neurokinin A after any of the steroid treatments than in rats treated with EB+progesterone (Fig. 3)(data not shown).

Posterior pituitaryHypothalamic peptide contentsInjection of EB did not significantly affect the substance PThe hypothalamic GnRH content 33 h after EB injection wascontent of the posterior pituitary (Fig. 3), while progesteronehigher than in oil-treated controls. In rats treated withinduced a decrease in content. Injection of EB+progesteroneEB+progesterone, the content was higher than in ratsalso induced a decrease compared to that after EB orinjected with progesterone alone (Fig. 1).progesterone injections. The neurokinin A content was notThe substance P content was higher 24 h after EB injectionsignificantly affected by any steroid treatment (data notthan in oil-treated controls, but was not significantly differentshown).from controls after 33 h. The content was lower in rats

treated with progesterone alone than in any other group 33 hafter the initial injection, while in rats treated withEB+progesterone, the content was higher than in rats Trigeminal nucleusinjected with progesterone alone (Fig. 2). However, content

The substance P content of the trigeminal nucleus wasin oil-treated rats was markedly higher 33 h after oil injectionincreased 33 h (but not 24 h) after injection of either EB orthan 24 h after oil injection.progesterone, and further increased in rats injected with bothThe neurokinin A content was higher in EB-injected ratssteroids. The neurokinin A content was not significantlythan in oil-injected rats at both studied times, whereasaffected by EB injection, but was increased after progesterone.progesterone had no significant effect. The content was higherThe content was lower in rats injected with EB+progesteroneafter injection of EB+progesterone than after progesteronethan in rats injected with progesterone alone (Fig. 4).alone (Fig. 2).

Anterior pituitary Cervical spinal cord

The substance P content of the cervical spinal cord was lowerInjection of EB had no significant effect on the substance Pcontent of the anterior pituitary, but the content was higher 24 h in EB injected rats than in oil-injected controls. 33 h

© 1998 Blackwell Science Ltd, Journal of Neuroendocrinology, 10, 823–828

Neurokinins and ovarian steroids 825

Time after EB injection

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F. 2. Hypothalamic substance P ( left panel ) and neurokinin A (right panel ) contents after vehicle (%), Estradiol benzoate (b), Progesterone (a),or Estradiol benzoate and Progesterone (q) treatment. Values were considered as different if P<0.005 (*).

after EB injection, the content was lower, but no different to oestrogen receptors. At the anterior pituitary, EB injectionhad no effect on the content of substance P or neurokinin A,oil-injected rats, or to rats injected with progesterone.

However, the content was higher in rats treated with while progesterone increased them, an action which wasabolished by the addition of EB. By contrast, other studiesEB+progesterone than in other groups at this time. The only

significant change in neurokinin A content was observed 24 h have reported that chronic treatment with EB down-regulatesthe substance P content of the anterior pituitary, but doesafter EB injection (Fig. 5).not affect the hypothalamic content (2, 22), but the differencein the duration of treatment may explain these differentDiscussionresults.

The regulation by ovarian steroids of substance P andIn the present study, the plasma concentration of substanceP was higher in EB-injected rats than in oil-injected controls neurokinin A in the hypothalamus and pituitary suggests

that neurokinins could be involved in the feed-back actionsonly during the phase of negative feed-back on LH secretion.During the first two days of the estrous cycle, the plasma of ovarian steroids on gonadotropin secretion. GnRH neur-

ones do not possess ovarian steroid receptors (23), but areconcentrations of E2 and substance P increase in parallel,suggesting a positive regulation of substance P secretion by regulated by other neurones, including substance P neurones,

which possess E2 receptors (24). Ovarian steroid regulationthe ovarian steroid (14).The changes in circulating concentrations of substance P of the substance P content in the hypothalamo-pituitary

complex appears to be due to regulation of the transcriptioncould reflect release from peripheral nerve terminals, orendocrine activity in the brain, the pituitary or the gastro- of the preprotachykinin-A gene encoding for substance P and

neurokinin A precursors (2).intestinal tract. By contrast with the effects of steroids onsubstance P concentrations, we detected no effects on plasma The precise relationship between the substance P and

neurokinin A contents in the trigeminal nucleus and spinalconcentrations of neurokinin A. This is surprising becauseboth neurokinins are encoded by the same gene (21), but it cord, and ovarian steroids, is not known. The present results

do not establish whether substance P and neurokinin Ais possible that EB and progesterone regulate enzymesresponsible for the cleavage of the precursor of neurokinins neurones are regulated by a direct action of ovarian steroids

on spinal nuclei or by an indirect signal from the hypothal-or the degradation of substance P and neurokinin A.In the hypothalamus, the content of substance P and amus. The changes in substance P and neurokinin A contents,

at both the hypothalamic and the spinal level, suggest changesneurokinin A appeared to be increased by EB at 24 h afterinjection. Furthermore, the present data indicate that proges- in the release of substance P and neurokinin A under E2and/or progesterone stimulation. It seems possible that theterone decreases the substance P content of the hypothalamus.Using an immunocytochemical method, Okamura et al. (15) sensitivity to nociceptive stimuli might vary with the ovarian

steroid environment, leading to a sexual dimorphism ofalso showed that a single EB injection increases the hypothal-amic substance P content, particularly in neurones containing nociceptive function.




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F. 4. Trigeminal nucleus substance P (upper part) and neurokinin A( lower part) contents after vehicle (%), Estradiol benzoate (b),Progesterone (a), or Estradiol benzoate and Progessterone (q) treat-ment. Values were considered as different if P<0.005 (*).

Materials and methods

Animals

Forty-two-day-old female Wistar rats (Charles River, Saint-Aubin-les-Elboeuf, France) were ovx under chloral hydrate anaesthesia (60 mg/rat).Fifteen days later, one group of rats was injected with sesame oil at 10.00 hon the first day (time 0) and killed 24 or 33 h later. A second group was


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injected with EB (50 mg/kg) and killed 24 or 33 h later. A third group wasF. 3. Posterior pituitary substance P (upper part), anterior pituitaryinjected with oil at time 0 and with progesterone (6 mg/kg) 24 h later, andsubstance P (middle part) and neurokinin A ( lower part) contents afterwere killed 33 h after time 0. The last group was injected with EB at time 0vehicle (%), Estradiol benzoate (b), Progesterone (a), or Estradioland with progesterone 24 h later, and were killed 33 h after the EB injection.benzote and Progesterone (q) treatment. Values were considered asThe plasma, hypothalamus, anterior and posterior pituitaries, the trigeminaldifferent if P<0.005 (*).nucleus and the cervical spinal cords were collected as previously described(14, 20).


Neurokinins and ovarian steroids 827

Assays

Plasma LH concentrations were assayed by a double antibody radioimmuno-logical assay (25). One hundred microliters of plasma were used for thedeterminations. Concentrations are expressed in ng calculated relative to ratLH RP-2 standard (NAIDDK, Bethesda, MD, USA). The sensitivity of theassay was 20 pg. GnRH was measured by a double antibody radioimmuno-logical assay (26). One percentage of the hypothalamus extract was used forthe determinations. Concentrations were expressed in ng calculated relativeto a synthetic peptide (Hoffman-Laroche, Basel, Switzerland). The assaysensitivity was 2 pg per assay tube. Substance P was measured by a doubleantibody radioimmumological assay (27) in 750 ml of plasma, 5% of thehypothalamic extract, 50% of the anterior or posterior pituitary extract, and2% of the trigeminal nucleus or cervical spinal cord extract. Concentrationswere expressed in pg calculated relative to a synthetic peptide (Neosystem,Strasbourg, France). The sensitivity of the assay was 2 pg. Neurokinin A wasmeasured by a double antibody radioimmunological assay (28) in 300 ml ofplasma, 5% of the hypothalamic extract, 10% of the anterior or posteriorpituitary extract, 1% of the trigeminal nucleus or cervical spinal cord extract.Concentrations were expressed in pg calculated relative to a synthetic peptide(Neosystem). The sensitivity of the assay was 2 pg. After an appropriatedilution, proteins were assayed according to the Bradford method (29) usingthe Bio Rad assay reagent (Bio Rad, Ivry sur Seine, France). The usedstandard was bovine serum albumin (Sigma) and contents were expressedin mg.

Statistical analysis

Values represent the mean±SEM of 7 plasma or tissue determinations. Thedata were analysed by an test followed by a posthoc Tukey’s test(SigmaStat, Jandel Scientific GmbH, Erkrath, Germany). Differences wereconsidered as significant for P<0.05.

Accepted 30 April 1998

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ovarian steroid modulation of neurokinin contents in hypothalamus, pituitary, trigeminal nucleus,...

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