Elżbieta Rębas

Central Opioid Inhibition of Neuroendocrine Stress Responses in Pregnancy in the Rat Is Induced by the Neurosteroid Allopregnanolone

The hypothalamus–pituitary–adrenal (HPA) axis is the major neuroendocrine stress response system. Corticotropin-releasing hormone (CRH) neurons in the parvocellular paraventricular nucleus (pPVN) play a key role in coordinating responses of this system to stressors. The cytokine interleukin-1β (IL-1β), mimicking infection, robustly activates these CRH neurons via a noradrenergic input arising from the nucleus tractus solitarii (NTS). In late pregnancy, HPA axis responses to stressors, including IL-1β, are attenuated by a central opioid mechanism that auto-inhibits noradrenaline release in the PVN. Here we show that the neuroactive progesterone metabolite allopregnanolone induces these changes in HPA responsiveness to IL-1β in pregnancy. In late pregnancy, inhibition of 5α-reductase (an allopregnanolone-synthesizing enzyme) with finasteride restored HPA axis responses (rapidly increased pPVN CRH mRNA expression, ACTH, and corticosterone secretion) to IL-1β. Conversely, allopregnanolone reduced HPA responses in virgin rats. In late pregnancy, activity of the allopregnanolone-synthesizing enzymes (5α-reductase and 3α-hydroxysteroid dehydrogenase) was increased in the hypothalamus as was mRNA expression in the NTS and PVN. Naloxone, an opioid antagonist, restores HPA axis responses to IL-1β in pregnancy but had no additional effect after finasteride, indicating a causal connection between allopregnanolone and the endogenous opioid mechanism. Indeed, allopregnanolone induced opioid inhibition over HPA responses to IL-1β in virgin rats. Furthermore, in virgin rats, allopregnanolone treatment increased, whereas in pregnant rats finasteride decreased proenkephalin-A mRNA expression in the NTS. Thus, in pregnancy, allopregnanolone induces opioid inhibition over HPA axis responses to immune challenge. This novel opioid-mediated mechanism of allopregnanolone action may alter regulation of other brain systems in pregnancy.

Effects of Somatostatin and Its Analogues on Tyrosine Kinase Activity in Rodent Tumors

The effects of native somatostatin-14 (SS-14) and of its two analogues, octreotide and CH-275, on the activity of tyrosine kinases (TK) in two rodent tumors (rat pituitary tumor and murine colonic cancer) were studied in vitro. The activity of TK was measured in tissue homogenates using γ[32P]ATP as the donor of the phosphoryl group and poly(Glu80, Tyr20) as a substrate. It was found that native SS-14 inhibited TK activity in both investigated tumors. Octreotide, which acts preferentially via somatostatin receptor subtype 2 (SSTR2), was very effective in inhibiting TK activity in the rat pituitary tumor, but it is a rather weak inhibitor of TK activity in murine colonic cancer. CH-275, a selective ligand of the SSTR1 subtype of SS receptors, suppressed TK activity in the pituitary tumor but was ineffective in the colonic cancer. It is hypothesized that the effect of neuropeptide somatostatin (SS-14) on murine colonic cancer is exerted via the subtype of receptor which does not interact with CH-275 and has no or low affinity for octreotide (SSTR 4, 3 or 5?).

Gender differences in steroid modulation of angiotensin II-induced protein kinase C activity in anterior pituitary of the rat.

To investigate whether the various steroid hormones can modulate the basal and angiotensin II-induced protein kinase C (PKC) activity in the anterior pituitary of the rat, female and male intact and ovariectomized female Wistar rats were treated in vivo with estradiol (E2), progesterone (P), dehydroepiandrostendione sulfate (DHEA-S), and pregnenolone sulfate (PREG-S). Estradiol caused the increase of basal PKC activity in intact and ovariectomized females, but did not change the enzyme activity in males. In ovariectomized animals the increase of PKC activity was lower than in intact females. Progesterone decreased PKC activity only in intact animals. DHEA-S strongly enhanced activity of PKC in ovariectomized females. Pregnenolone sulfate did not significantly change PKC function of all studied groups. Incubation with AngII enhanced the PKC activity in intact (without steroid treatment) animals of both genders. In females, AngII and estradiol together rise the PKC-stimulated phosphorylation in greater degree than used separately. Treatment with other investigated steroids reduced the effect of AngII. In intact males every examined hormone turned back the stimulatory effect of AngII on PKC activity. These data suggest that gender differences in PKC activity are likely related to hormonal milieu of experimental animals and may depend in part on the basic plasma level of estrogens.

The effect of angiotensin III on protein tyrosine kinase activity in rat pituitary

Angiotensin III is the biological active peptide from the angiotensin family, which play the important role in several cellular processes. Ang III is the product of reaction catalyzed by aminopeptidase A and in turn can be a substrate for aminopeptidase N, enzyme which converts Ang III to shorter fragment, Ang IV. Aminopeptidase N is specifically inhibited by PC18. Ang III can act by binding to receptors AT1, AT2 or other type of receptors, which are not well recognized. The connection of Ang III to AT1 and AT2 receptors could be inhibited by losartan or PD123319, respectively. The aim of this study was to investigate what is the influence of angiotensin III on protein tyrosine kinase activity in rat pituitary and what is the possible place of interaction of Ang III with target cells. The obtained results show that Ang III can modify tyrosine kinase activity in concentration dependent manner but Ang IV appeared more effective. In presence of PC18 Ang III caused the same changes as Ang III alone that suggests that Ang III, not its metabolite modify tyrosine kinase activity. Losartan and PD123319 given together with Ang III enhanced the changes induced by Ang III. It suggests that the investigated peptide has an effect on protein tyrosine kinase activity in a different way than via AT1 and AT2 receptors.

GABA-shunt enzymes activity in GH3 cells with reduced level of PMCA2 or PMCA3 isoform.

GABA (γ-aminobutyric acid) is important neurotransmitter and regulator of endocrine functions. Its metabolism involves three enzymes: glutamate decarboxylase (GAD65 and GAD67), GABA aminotransferase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). As many cellular processes GABA turnover can depend on calcium homeostasis, which is maintained by plasma membrane calcium ATPases (PMCAs). In excitable cells PMCA2 and PMCA3 isoforms are particularly important. In this study we focused on GABA-metabolizing enzymes expression and activity in rat anterior pituitary GH3 cells with suppressed expression of PMCA2 or PMCA3. We observed that PMCA3-reduced cells have increased GAD65 expression. Suppression of PMCA2 caused a decrease in total GAD and GABA-T activity. These results indicate that PMCA2 and PMCA3 presence may be an important regulatory factor in GABA metabolism. Results suggest that PMCA2 and PMCA3 function is rather related to regulation of GABA synthesis and degradation than supplying cells with metabolites, which can be potentially energetic source.

Calcium-engaged Mechanisms of Nongenomic Action of Neurosteroids

Background: Neurosteroids form the unique group because of their dual mechanism of action. Classically, they bind to specific intracellular and/or nuclear receptors, and next modify genes transcription. Another mode of action is linked with the rapid effects induced at the plasma membrane level within seconds or milliseconds. The key molecules in neurotransmission are calcium ions, thereby we focus on the recent advances in understanding of complex signaling crosstalk between action of neurosteroids and calcium-engaged events. Methods: Short-time effects of neurosteroids action have been reviewed for GABAA receptor complex, glycine receptor, NMDA receptor, AMPA receptor, G protein-coupled receptors and sigma-1 receptor, as well as for several membrane ion channels and plasma membrane enzymes, based on available published research. Results: The physiological relevance of neurosteroids results from the fact that they can be synthesized and accumulated in the central nervous system, independently from peripheral sources. Fast action of neurosteroids is a prerequisite for genomic effects and these early events can significantly modify intracellular downstream signaling pathways. Since they may exert either positive or negative effects on calcium homeostasis, their role in monitoring of spatio-temporal Ca2+ dynamics, and subsequently, Ca2+-dependent physiological processes or initiation of pathological events, is evident. Conclusion: Neurosteroids and calcium appear to be the integrated elements of signaling systems in neuronal cells under physiological and pathological conditions. A better understanding of cellular and molecular mechanisms of nongenomic, calcium-engaged neurosteroids action could open new ways for therapeutic interventions aimed to restore neuronal function in many neurological and psychiatric diseases.