Long term exposure to dopamine reverses the inhibitory effect of endothelin-1 on prolactin secretion.

This study was undertaken to assess the possibility that endothelin-1 (ET-1) and dopamine (DA) can act in concert to modulate PRL secretion. Enzymatically dispersed anterior pituitary cells obtained from random cycling female rats were perifused with Dulbecco's Modified Eagle's Medium supplemented with 0.2% BSA and 100 microM ascorbic acid. In the absence of dopamine, ET-1 (applied at 20 nM for 60 min) rapidly evoked a small transient elevation of PRL release, followed by a sustained inhibitory phase. Overnight perfusion with 500 nM DA-supplemented medium did not change the basic character of ET-1's effects on PRL secretion. Continuation of DA exposure for 48 h dramatically shifted the responsiveness of the lactotrophs to ET-1; the fast stimulatory response was robustly enhanced, whereas the inhibitory phase was replaced by a modest secondary elevation of basal PRL secretion. The stimulatory effect of ET-1 on PRL secretion after DA pretreatment was blocked by an ETA receptor antagonist, BQ-123. The effect of DA can be mimicked completely by a specific D2 receptor agonist (+/-)-2-(N-phenyl-N-propyl)amino-5-hydroxytetraline hydrochloride, whereas pretreatment with a D1 agonist, SKF-39393, failed to change the responsiveness of lactotrophs to ET-1. Our data indicate that persistent activation of D2 receptors, a condition most closely resembling the in vivo environment of the lactotrophs, uncouples the inhibitory signaling pathway from the ETA receptor while synergistically affecting signal transduction, which mediates the ET-induced stimulation of PRL secretion.

The effects of endothelins on the secretion of prolactin, luteinizing hormone, and follicle-stimulating hormone are mediated by different guanine nucleotide-binding proteins.

Different bacterial toxins capable of modifying specific alpha-subunits of G-proteins were used to characterize the guanine nucleotide-binding protein (G-protein) dependency of the effects of endothelins (ETs) on PRL, LH, and FSH secretion. Primary cultures of anterior pituitary cells obtained from female rats were preincubated for 24 h with 20 ng/ml pertussis toxin (PTX) or 2 micrograms/ml cholera toxin (CTX) before challenge with ETs. Both ET-1 and ET-3 elicited a concentration-dependent inhibition of PRL secretion and stimulated the release of LH and FSH secretion on pituitary cells not treated with toxins. Based on the calculated ration of the half-maximal effective concentrations (EC50) of ET-1 and ET-3, ET-1 showed 7800, 20, and 14 times greater potency than ET-3 on PRL, LH, and FSH secretion, respectively. PTX, a selective inhibitor of Gi and several other G proteins, increased the basal secretion of PRL and completely eliminated the responsiveness of lactotroph cells to ET-1 and ET-3. Pretreatment with PTX caused a markedly different effect on LH and FSH secretion: while basal LH release was slightly increased, FSH secretion was markedly depressed by PTX. Moreover, while ET-induced LH secretion was enhanced by PTX, the effectiveness of ETs on FSH release was completely abolished. CTX, known as an activator of Gs proteins, decreased the basal secretory activity of lactotrophs but did not influence the ET-induced decrease of PRL release. CTX pretreatment (like PTX before) elicited a strikingly different effect on LH and FSH: while basal LH secretion was enhanced, basal FSH secretion was markedly inhibited by CTX. Moreover, while the effectiveness of ETs on LH secretion was not changed significantly, the stimulatory effect of ETs on FSH secretion was diminished after CTX pretreatment. Thus, the inhibition of PRL secretion by ETs requires a PTX-sensitive G protein while the ET-induced stimulation of FSH secretion involves both PTX- and CTX-sensitive elements. The fact that pretreatments with PTX or CTX influenced basal secretion of PRL, LH, and FSH suggests that PTX- and/or CTX-sensitive G proteins are directly involved in the process of exocytosis. Additionally, these findings might indicate an active paracrine/autocrine regulation of pituitary cells in culture that are impaired or enhanced by the bacterial toxins employed. Though the broad substrate specificity of PTX and CTX and the multiplicity of G protein families did not allow us to identify the specific G protein(s) involved, these data reveal the diversity of ET-induced intracellular signaling mechanisms in lactotrophs and gonadotrophs.

The hypothalamic paraventricular nucleus has a pivotal role in regulation of prolactin release in lactating rats.

The affect of paraventricular nucleus (PVN) lesions on PRL secretory response to suckling was studied in adult female rats. Basal levels of PRL were similar in the control and lesioned groups. Substantial decreases in PRL levels occurred after separation of pups from their mothers in the control as well as lesioned animals. When mothers and pups were reunited, the circulating PRL concentrations of the control groups rose immediately from basal values of 50-100 micrograms/liter to reach peaks of 450-550 micrograms/liter. PVN lesions significantly decreased the suckling-induced rise of PRL levels. Furthermore, PVN lesions abolished the high amplitude, episodic pattern of PRL release in continuously lactating rats. These findings are consistent with the view that PVN neurons produce PRL releasing factor(s), which is (are) required for normal secretory patterns of PRL in lactating rats.

Endothelin activates large-conductance K+ channels in rat lactotrophs: reversal by long-term exposure to dopamine agonist.

Endothelin-1 (ET-1) inhibits PRL secretion from cultured rat lactotrophs. However, ET-1 stimulates PRL secretion after cultured lactotrophs have been exposed for 48 h to dopamine or D2 dopamine agonists. In the present study, we have used cell-attached and inside-out patch recordings to establish an ionic basis for these effects. Bath application of 20 nM ET-1 to untreated lactotrophs evoked a robust and persistent activation of large-conductance K+ channels in cell-attached patches. This effect of ET-1 had a long latency to onset, was maintained for as long as ET-1 was present, and required at least 10 min of washing in control saline before complete recovery was achieved. The stimulatory effect of 20 nM ET-1 on these channels was markedly attenuated in the presence of the selective ET(A) receptor antagonist BQ-610 (200 nM), or after pertussis toxin (200 ng/ml, 16 h) pretreatment. The unitary slope conductance of the ET-1 activated channels in cell attached patches was 165 and 95 pS when the recording electrodes contained 150 and 5.4 mM KCl, respectively. These channels were voltage-sensitive and their activity increased upon patch depolarization. Previously activated channels in cell-attached patches became quiescent immediately upon patch excision into Ca2+-free bath saline. Exposure of the intracellular surface to 0.1 microM Ca2+ restored the activity of these channels similar to the level seen before patch excision. In addition, preincubating the cells with the membrane-permeable Ca2+-chelator BAPTA-AM, or using Ca2+-free solution in the recording pipettes, prevented the activation of these channels by ET-1. The ET-1 activated large-conductance Ca2+-dependent K+ (BK(Ca)) channels were blocked by 20 mM tetraethylammonium but were insensitive to the K+ channel blockers apamin (1 microM), charybdotoxin (200 nM), or iberiotoxin (200 nM). Acute application of 10 microM dopamine and 20 nM ET-1 caused activation of BK(Ca) channels with indistinguishable kinetic properties, although the effect of dopamine occurred with shorter latency. After 48-h exposure to the specific D2 dopamine receptor agonist (+/-)-2-(N-phenyl-N-propyl) amino-5-hydroxytetralin hydrochloride (PPHT, 500 nM), bath application of 20 nM ET-1 resulted in inhibition of spontaneously active BK(Ca) channels. These data suggest that both the stimulatory and inhibitory effects of ET-1 on PRL secretion are mediated, at least in part, by actions on BK(Ca) channels, and that long term exposure to dopamine or D2 agonists alters the signaling pathways from the ET(A) receptor to BK(Ca) channels.

Endothelin is an autocrine regulator of prolactin secretion.

The aim of this study was to establish the cellular source of ET-like peptides affecting PRL secretion. Fluorescence double label immunocytochemistry and confocal laser scanning microscopy were used to demonstrate cellular colocalization for PRL and endothelin-1 (ET1)-like immunoreactivities in the anterior lobe of the pituitary gland of rats. An ET-specific reverse hemolytic plaque assay was applied to demonstrate that lactotrophs are capable of releasing ET-like peptides. A PRL-specific reverse hemolytic plaque assay was used to assess the influence of the released endogenous ETs on PRL secretion. ET(A)-specific receptor antagonists BQ123 and BQ610, and endothelin convertase enzyme inhibitory peptide, [22Val]big ET1-(16-38), increased PRL secretion, whereas the ET(B) receptor-specific antagonist BQ788 was ineffective. The ET(A) antagonist BQ123-induced increase in PRL secretion followed a bell-shaped dose-response curve in cells obtained from female rats, whereas it followed a sigmoid curve in males. Frequency distribution of PRL plaque sizes using logarithmically binned data revealed two subpopulations of lactotrophs with differential responsiveness to endogenous ETs. These data demonstrate that a large proportion of lactotrophs is capable of expressing and secreting ET-like peptides in biologically significant quantities. As low pituitary cell density in reverse hemolytic plaque assay minimizes cell to cell communications, these findings constitute direct proof of autocrine regulation of PRL secretion by ET-like peptides.

Hysterectomy-induced alterations in prolactin secretion of lactating rats.

The contribution of the uterus to the regulation of PRL secretion in lactating dams and cycling female rats was investigated. Lactating animals were hysterectomized or sham operated 2 days after parturition, and the number of pups was adjusted to eight. Blood samples for PRL RIA were obtained through intra-atrial cannulae implanted 2 days before experimentation. In order to study the PRL secretory profile in undisturbed freely lactating rats, blood samples were taken every 2 h for 24 h starting at 1400 h. During early lactation (days 7-8), hysterectomy did not alter the PRL secretory profile compared to that of sham-operated controls. On days 14-15 post partum, PRL secretion followed a characteristic bimodal pattern showing two PRL surges at 1800 h and 0600 h. After hysterectomy, the early morning PRL surge disappeared and PRL secretion showed an unimodal daily rhythm reaching its peak at 1800 h. The possible effect of the absence of the uterus on suckling-induced PRL release at various stages of lactation was studied. On days 7-8, suckling stimuli after 4 h of pup deprivation induced robust PRL release. Hysterectomy did not significantly alter PRL release at this earlier stage of lactation. In control groups, the suckling-induced PRL secretory response markedly declined as the postpartum period advanced. On the other hand, the hysterectomized animals retained significantly greater responsiveness to suckling during the second half of lactation. These data indicate an inhibitory influence of the uterus on PRL secretion. The onset of this uterine effect is considerably delayed, and its influence became prominent only at a later phase of lactation. The effect of length of pup deprivation preceding the suckling stimulus, in combination with hysterectomy, was also investigated. Hysterectomy significantly increased suckling-induced PRL release after 4 and 24 h separation compared to the sham-hysterectomized animals. When the separation was longer than 48 h, the inducibility of PRL release by suckling declined and was not influenced by hysterectomy. In order to study the possible influence of the uterus on PRL secretion during the estrous cycle, regularly cycling female rats were hysterectomized at diestrus 1. Twelve days later the animals were cannulated, and serial blood samples were taken during the subsequent proestrus. Hysterectomy did not alter the PRL surge which occurred on the afternoon of proestrus indicating that the uterus does not have a major function in regulating PRL secretion on proestrus. In conclusion, hysterectomy significantly delayed the extinction of suckling-induced PRL release revealing the active role of the uterus in the regulation of this neuroendocrine reflex.

Effects of naloxone and neonatal treatment with monosodium-L-glutamate on growth hormone and prolactin release induced by electrical stimulation of the medial-basal hypothalamus in rats.

The role of nerve cells of the arcuate nucleus and endogenous opioid peptides in the regulation of GH and prolactin secretion has been investigated. Electrical stimulation of the medial-basal hypothalamus (MBH) for 10 min raised plasma levels of both hormones in male rats anaesthetized with pentobarbitone sodium. Plasma hormone levels increased within 5 min after the termination of the stimulus, while no marked changes were found during stimulation. The GH response to the electrical stimulus was substantially reduced in rats with arcuate lesions induced by neonatal treatment with monosodium-L-glutamate (MSG). By contrast, the size of the prolactin response was not altered by MSG treatment. The opiate receptor antagonist naloxone (10 mg/kg, i.v.) failed to influence GH secretion induced by electrical stimulation in either control or MSG-treated animals. The post-stimulus rise of plasma prolactin levels was attenuated by naloxone in control rats, while the same dose of the drug was ineffective in rats which had been exposed to MSG. We conclude that endogenous opioids participate in the increase of prolactin release upon electrical stimulation of the MBH but are not involved in the GH secretory response. Arcuate neurones are important in the maintenance of the GH response to electrical stimulation. By contrast, lesioning of the arcuate nucleus failed to affect the prolactin secretory response elicited by MBH stimulation. However, prolactin release in MSG-treated rats appeared less susceptible to the inhibitory action of naloxone, suggesting a possible supersensitivity towards endogenous opioids.

Decrease of morphine-induced prolactin release by a procedure causing prolonged stress.

The effects of morphine and fentanyl on plasma prolactin levels in rats have been measured. It was found that a prolonged immobilization stressful procedure for 5 h inhibited the response to morphine and fentanyl to increase prolactin secretion, but did not influence the increase in plasma prolactin caused by haloperidol. The injection of a large dose of cortisol (25 mg/kg, s.c.) also evoked an inhibition of morphine-induced prolactin release. The inhibition was maximal 24 h after the administration of the glucocorticoid. These results indicate that stress may induce prolonged alteration in endogenous opioid-mediated neuromodulation via a prolonged release of glucocorticoids.

Effect of chronic morphine treatment on the adrenaline biosynthesis in adrenals and brain regions of the rat.

Phenylethanolamine-N-methyltransferase (PNMT) activity and tissue catecholamine content were examined after 13 days morphine treatment. Prolonged morphine treatment did not alter the PNMT activity in brain regions (A1/C1 and A2/C2 cell groups, medial basal hypothalamus, median eminence). However, the enzyme activity and the adrenaline content were increased in the adrenal medulla of male rats. In parallel, morphine treatment induced adrenal hypertrophy. In female or hypophysectomized male animals the chronic morphine treatment had no effect on adrenal weight but evoked the increase of PNMT activity. It is concluded that the morphine-induced increased adrenaline biosynthesis in the adrenal gland is not fully dependent on the intact pituitary-adrenal axis and may be mediated partly by a neural mechanism (increased splanchnic nerve activity) or by a direct effect of morphine.

Nuclear translocation of STAT5 and increased expression of Fos related antigens (FRAs) in hypothalamic dopaminergic neurons after prolactin administration.

Ample evidence indicates feedback relationships between pituitary prolactin and hypothalamic dopaminergic neurons. Since the presence of prolactin receptors was earlier demonstrated in hypothalamic dopaminergic neurons, our working hypothesis was that prolactin induced activation of prolactin receptor coupled signaling leads to increased neuronal activity in these neurons. The aim of this study was to correlate prolactin receptor mediated signaling and prolactin induced activation in hypothalamic dopaminergic neurons. We used nuclear translocation of STAT5 as a marker of prolactin receptor induced signaling and expression of Fos related antigens (FRAs) as an indicator of neuronal activation. We performed double label immunocytochemical studies to determine the time course of the presence of FRAs and STAT5 in the nuclei of hypothalamic dopaminergic neurons after ovine prolactin treatment. Exogenous ovine prolactin treatment of ovariectomized rats resulted in an increase in serum ovine prolactin levels and a decrease in endogenous serum prolactin levels, indicating that ovine prolactin activated mechanisms inhibited pituitary prolactin secretion. Indeed, ovine prolactin activated the prolactin receptors in most subpopulations of hypothalamic dopaminergic neurons, resulting in nuclear translocation of STAT5. Also, increased neuronal activity, indicated by expression of FRAs, was observed in the same neuron populations after ovine prolactin treatment. These results suggest that signal transduction mechanisms coupled to prolactin receptors in hypothalamic dopaminergic neurons resemble those observed in other tissues; and nuclear translocation of STAT5 can be used as a marker of prolactin receptor activation in hypothalamic dopaminergic neurons.

Noradrenergic innervation of the rat hypothalamus:experimental biochemical and electron microscopic studies.

The concentrations of noradrenaline in individual hypothalamic nuclei and in the median eminence were measured 7-10 days following surgical transections of the lower brain stem or electrolytic lesions of the medullary noradrenaline-containing cell groups. Terminal degeneration in the hypothalamus was studied after the same surgical procedures. Direct, monosynaptic connections between the dorsomedial hypothalamic nucleus and all the noradrenaline-containing cell groups investigated were found. Degenerated synaptic boutons were demonstrated in the median eminence, arcuate, dorsomedial, ventromedial, periventricular and paraventricular nuclei following lesions of the solitary tract and of the lateral reticular nucleus. Biochemical measurements indicate that the pontine-medullary noradrenergic cell groups are the source of hypothalamic norepinephrine. Ascending noradrenergic fibers destined to terminate in the hypothalamus are provided by several cell groups, though the bulk of the NA-fibers seem to originate in the A1-group, in the ventrolateral part of the medullary reticular formation. Most of these fibers join the ventral NA bundle, fewer join the dorsal periventricular tract and several probably also join the dorsal NA bundle. A significant overlap was found in the hypothalamic arborization of the noradrenergic fibers, so that no strict topographical organization seems to be present either in their origin or their termination.

Inhibition of prolactin secretion by endothelin-3 is pertussis toxin-sensitive.

The effect of pertussis toxin (PTX) pretreatment on endothelin-3 (ET-3)-mediated inhibition of prolactin secretion from primary cultures of rat anterior pituitary cells was examined. Monolayer cultures of anterior pituitary cells were treated with either 20 ng/ml PTX dissolved in media or with media alone (control) on the third day of culture. Exactly 24 h after PTX pretreatment, cells were challenged with either 100 nM ET-3 dissolved in media or media alone (control) for 4 or 48 h. ET-3 significantly (P less than 0.01) inhibited prolactin secretion in both the 4 and 48 h incubations. However, if the cells had been previously treated with PTX, ET-3 did not significantly affect prolactin secretion. These data suggest that a PTX-sensitive G protein mediates ET-3-induced inhibition of prolactin secretion and that ET-3 may invoke a signal transduction mechanism in the lactotroph which is distinct from those described in other cell types.

Effects of beta-adrenergic inhibitors on noradrenaline and adrenaline metabolism in brain regions of the rat.

Chloranolol (5 mg/kg i.p.) retarded the disappearance of noradrenaline induced by the dopamine-beta-hydroxylase (DBH) inhibitor FLA 63, in the hypothalamus, nucleus of the solitary tract (A-2/C-2 area), and lateral reticular nucleus (A-1/C-1 area) regions, while propranolol (20 mg/kg i.p.) was effective only in the hypothalamus and in the lateral reticular nucleus. The DBH inhibitor-caused accumulation of dopamine was also inhibited by chloranolol in the solitary tract nucleus. Chloranolol and propranolol were able to antagonize the fall in adrenaline concentration due to the phenylethanolamine-N-methyltransferase (PNMT) inhibitor, Lilly 87130, only in the region of the solitary tract nucleus. The data suggest that beta-adrenergic inhibitors reduce noradrenaline and adrenaline turnover in the central nervous system, most characteristically in the solitary tract nucleus and that this effect is possibly related to their antihypertensive effectiveness.

Effects of reserpine and antidepressants on dopamine and DOPAC (3,4-dihydroxyphenylacetic acid) concentrations in the striatum, olfactory tubercle and median eminence of rats.

The dopamine, noradrenaline and 3,4-dihydroxyphenylacetic acid (DOPAC) levels of various rat brain areas (median emience, olfactory tubercle, rostral and caudal part of the striatum) were measured. The effects of reserpine and antidepressants: amitriptyline, nomifensine and EGYT 475 (1-benzyl-4-/2'-piridylcarbonyl/piperazine) were investigated. The dopamine content of the median eminence proved to be the most sensitive to the depletory effect of reserpine. Reserpine (1 mg/kg i.p.) caused a decrease of dopamine in the median eminence, in the olfactory tubercle and in the rostral striatum but not in the caudal striatum. Amitriptyline and EGYT 475 antagonized the reserpine-induced dopamine depletion in the olfactory tubercle. In the median eminence amitriptyline attenuated the effect of the depletor while nomifensine augmented it; EGYT 475 was without effect. The antidepressants antagonized the reserpine-induced increase of DOPAC in the rostral part of the striatum.

Catecholamine concentration of various brain nuclei of the rat as affected by ACTH and corticosterone.

The acute effect of ACTH and corticosterone on catecholamine concentration of individual brain nuclei has been examined. ACTH significantly increased the noradrenaline (NA) content in the locus coeruleus, and decreased it in the ventromedial and arcuate nuclei. No change was observed after ACTH in the dopamine (DA) concentrations of the mentioned areas and in the catecholamine level of the mamillary body, hippocampus, medial habenular nucleus and of the pituitary. Corticosterone increased the NA content of the supraoptic nucleus and the NA and DA levels in the median eminence, while it did not affect the amine concentrations in the locus coeruleus.

Adrenergic innervation of the rat hypothalamus.

The adrenergic innervation of the hypothalamus was studied by measuring hypothalamic adrenaline levels following surgical transection of the lower brain stem or electrolytic lesion of the medullary adrenaline-containing cell groups. The adrenaline levels in some hypothalamic nuclei and in the median eminence showed a slight decrease after partial transection of the medulla oblongata, whilst there was a pronounced decrease (by 59-78%) 7-10 days following total hemisection or unilateral lesion of the C1-catecholaminergic cell group in the medulla oblongata.

Distribution of norepinephrine and dopamine in cerebral cortical areas of the rat.

Concentrations of norepinephrine and dopamine were determined using enzyme isotope assay in 27 microdissected cerebral cortical areas of the rat. A detailed map is presented for microdissection of rat cerebral cortex. Norepinephrine was found in low but still measurable quantities throughout the cortex. Differences between cortical areas are also low. Relatively highest levels were demonstrated in the pyriform, insular and entorhinal cortices. The distribution of dopamine was found to be uneven with a maximal regional difference of 1:24. Concentration of dopamine was in all areas lower than that of norepinephrine. The highest dopamine concentration (2,4 ng/mg protein) was measured in the rostral pyriform cortex but other mesocortical (cingulate, frontal, insular and entorhinal) dopaminergic areas also contained relatively high amounts. Except for the caudal occipital and caudal entorhinal cortices all regions studied contained measurable quantities of dopamine. Its low concentration relative to norepinephrine (below 15%) suggests that in the cortical areas studied dopamine is present as the precursor of norepinephrine.

Long-term ACTH induced diminished responsiveness of prolactin secretion to morphine.

The effect of morphine on plasma prolactin level and on dopamine turnover in the median eminence was studied using adult male rats chronically treated with ACTH. It was found that the ACTH pretreatment caused a decrease in the effect of morphine on prolactin secretion and prevented the inhibitory effect of morphine on dopamine turnover measured in the median eminence. The prolonged ACTH administration did not influence the prolactin content of the pituitaries and the in vitro dopamine sensitivity of lactotroph cells. Acute dexamethasone injection did not change the morphine-caused prolactin release. These results suggest that chronic ACTH treatment (possibly via corticosterone hyperproduction) elicits an opiate-tolerance like state of tuberoinfundibular dopaminergic neurons.

Loss of sensitivity to morphine induced by prolonged ACTH treatment.

The effect of long term ACTH treatment on some actions of morphine were studied. The effect of ACTH administration was compared to that induced by acute dexamethasone injection. ACTH caused a delayed inhibition of the morphine induced increase in growth hormone secretion demonstrable 24 hr after the last hormone injection. The morphine induced increase of striatal DOPAC (3,4-dihydroxyphenylacetic acid) content was also inhibited by ACTH treatment, however, neither the analgesia, nor the hypermotility caused by morphine were affected. Dexamethasone did not alter significantly the responsiveness to morphine. It is concluded that the prolonged exposure to ACTH presumably causes a corticosterone-mediated loss of responsiveness of functionally restricted opiate sensitive mechanisms in the central nervous system.