N. S. Bondarenko

Signal molecules during the organism development: Central and peripheral sources of noradrenaline in rat ontogenesis.

Using the method of high performance liquid chromatography with electrochemical detection, the age dynamics of the content of noradrenaline (NA) in the brain, adrenal gland, and the organ of Zuckerkandl in prenatal (18th and 21st days of embryogenesis) and early postnatal (3, 7, 15, and 30th days) periods of development was studied. The potential contribution of these organs to the formation of physiologically active concentration of noradrenalin in the blood was also assessed. The results suggest that, during the development of the organism, the activity of the sources of noradrenaline in the general circulation changes, which gives a reason to assume the existence of humoral interaction between NA-producing organs in the perinatal period of ontogenesis.

The secretion of noradrenaline from the brain into the peripheral blood during rat ontogenesis

We studied the endocrine function of the noradrenergic system in the developing brain. The data on the age-related dynamics of the noradrenaline concentration in plasma and in the hypothalamus and mesencephalon–rhombencephalon before and after the formation of the blood–brain barrier indirectly indicates the possibility of noradrenaline secretion from the brain to the peripheral blood. The direct evidence that the rat brain may be a source of noradrenaline in the peripheral blood before the formation of the blood–brain barrier was first obtained using a model of chronic specific inhibition of noradrenaline synthesis in the neonatal rat brain. It suggests that the brain is involved in the regulation of the development and functioning of peripheral target organs during this period.

Modeling of chronic selective inhibition of noradrenaline synthesis in the brain of neonatal rats

123 Ontogenetic development and functioning of an organism are regulated by intracellular chemical sig naling; the signal molecules are secreted by the neu roendocrine system and function as morphogenetic or transcription factors affecting target cells and organs via specific receptors. Among the great variety of chemical signals controlling the development of target organs and tissues, noradrenaline (NA), which is syn thesized mainly by brain neurons, peripheral sym pathoadrenal system, and extra adrenal chromaffine tissue, is one of the most efficient factors exhibiting a broad range of physiological effects [1, 2]. We have proposed a hypothesis that, before the for mation of the blood–brain barrier (BBB), the brain functions as an endocrine organ secreting physiologi cally active substances into the general circulation and thus has a direct endocrine effect on the develop ment and functioning of peripheral target organs and tissues [3]. Evidence efficiently supporting this hypothesis was obtained in our previous study using serotonin, dopamine (DA), and gonadotropin releasing hor mone as markers of endocrine functions of the devel oping brain [4]. The purpose of the present work was to obtain direct evidence in support of our hypothesis that the developing brain functions as an endocrine source secreting NA into the general circulation. In particu lar, our aim was to develop a model of chronic (irre versible) suppression of NA synthesis by brain neurons in ontogeny before the BBB formation and to evaluate the changes in NA levels in the brain, peripheral blood, and peripheral organs using this model. The study was performed in male Wistar rats ana lyzed on postnatal days 2, 3, and 6 (P2, P3, and P6). The day when animals were born was considered post natal day 1. Animals were kept in a vivarium under standard conditions with unlimited food and water supply. Long term (chronic) pharmacological inhibition of NA synthesis in rat brain was ensured by adminis tration of 6 hydroxydopamine (6 OHDA), a specific neurotoxin that causes degeneration of catecholamin ergic neurons. Via DA and NA membrane transport ers, 6 OHDA is transported into the target cells and induces uncoupling of the oxidative phosphorylation chain and oxidative stress in mitochondria, which results in cell death [5]. GBR 12909, which inhibits 6 OHDA reuptake by DA neurons and thus acts as a specific neuroprotector, was administered systemi cally 60 min prior to 6 OHDA injections to prevent the death of dopaminergic neurons (DA neurons) [6].

Plasticity of central and peripheral sources of noradrenaline in rats during ontogenesis

The morphogenesis of individual organs and the whole organism occurs under the control of intercellular chemical signals mainly during the perinatal period of ontogenesis in rodents. In this study, we tested our hypothesis that the biologically active concentration of noradrenaline (NA) in blood in perinatal ontogenesis of rats is maintained due to humoral interaction between its central and peripheral sources based on their plasticity. As one of the mechanisms of plasticity, we examined changes in the secretory activity (spontaneous and stimulated release of NA) of NA-producing organs under deficiency of its synthesis in the brain. The destruction of NA-ergic neurons was provoked by administration of a hybrid molecular complex–antibodies against dopamine-β-hydroxylase associated with the cytotoxin saporin–into the lateral cerebral ventricles of neonatal rats. We found that 72 h after the inhibition of NA synthesis in the brain, its spontaneous release from hypothalamus increased, which was most likely due to a compensatory increase of NA secretion from surviving neurons and can be considered as one of the mechanisms of neuroplasticity aimed at the maintenance of its physiological concentration in peripheral blood. Noradrenaline secretion from peripheral sources (adrenal glands and the organ of Zuckerkandl) also showed a compensatory increase in this model. Thus, during the critical period of morphogenesis, the brain is integrated into the system of NA-producing organs and participates in their reciprocal humoral regulation as manifested in compensatory enhancement of NA secretion in each of the studied sources of NA under specific inhibition of NA production in the brain.

Reciprocal humoral regulation of endocrine noradrenaline sources in perinatal development of rats

The goal of the present study was to verify our hypothesis of humoral interaction between the norepinephrine secreting organs in the perinatal period of ontogenesis that is aimed at the sustaining of physiologically active concentration of norepinephrine in blood. The objects of the study were the transitory organs, such as brain, organ of Zuckerkandl, and adrenals, the permanent endocrine organ of rats that releases norepinephrine into the bloodstream. To reach this goal, we assessed the adrenal secretory activity (norepinephrine level) and activity of the Zuckerkandl’s organ under the conditions of destructed noradrenergic neurons of brain caused by (1) their selective death induced by introduction of a hybrid molecular complex, which consisted of antibodies against dopamine-β-hydroxylase (DBH) conjugated with saporin cytotoxin (anti-DBH-saporin) into the lateral brain ventricles of neonatal rats; and (2) microsurgical in utero destruction of embryo’s brain (in utero encephalectomy). It was observed that 72 h after either pharmacological or microsurgical norepinephrine synthesis deprivation in the newborn rat’s brain, the level of norepinephrine was increased in adrenals and, conversely, decreased in the Zuckerkandl’s organ. Therefore, the experiments with models of chronical inhibition of norepinephrine synthesis in prenatal and early postnatal rat’s brain revealed changes in the secretory activity of peripheral norepinephrine sources. This, apparently, favors the sustaining of physiologically active norepinephrine level in the bloodstream.

Secretory activity of the brain and peripheral organs: Spontaneous and stimulated release of noradrenaline in the ontogenesis of rats

Spontaneous and K+-stimulated release of noradrenaline from the hypothalamus, adrenal gland, and organ of Zuckerkandl under their flowing incubation was investigated in the perinatal period of ontogenesis of rats. The results suggest that, during the investigated period of ontogenesis, adrenal glands are the main source of noradrenaline in the blood, whereas the contributions of the organ of Zuckerkandl and the brain are not as significant and change during this period.

Effect of Prolyl-Glycyl-Proline (PGP) and Its Acetylated Form (N-AcPGP) on Calcium Level in the Cytoplasm of Rat Peritoneal Mast Cells.

Tripeptide glycyl-prolyl-proline (PGP), a regulatory peptide of the glyproline family, possesses a pronounced anti-inflammatory effect primarily due to its ability to prevent secretion of the proinflammatory mediator histamine by rat peritoneal mast cells. Activation of mast cell with synacthen (ACTH1-24) and substance 48/80 leads to an increase in intracellular calcium concentration. Pretreatment of mast cells with PGP prevented calcium entry into the cytoplasm from both intercellular space and intracellular stores. Acetylated peptide (N-AcPGP) produced a similar effect on histamine release and intracellular calcium content in mast cells activated with synacthen. These findings indicate that both forms of the peptide can stabilize mast cells and prevent intracellular calcium increase.

Gene expression and the contents of noradrenaline synthesis enzymes in the rat brain during the critical period of morphogenesis

The aim of this study was to investigate the molecular-genetic mechanisms of noradrenaline synthesis in the rat brain during the critical period of morphogenesis. For this purpose, the level of gene expression and the contents of the enzymes for noradrenaline (NA) synthesis, tyrosine hydroxylase (TH), and dopamine-β-hydroxylase were evaluated. We have shown that the mRNA level of these enzymes remains at a stable high level in the perinatal period of ontogeny and decreases slightly by the end of the first month of life. The contents of these enzymes in the brain have a different age dynamics: the content of TH, a rate-limiting enzyme of NA synthesis, is constantly growing, which, along with the constant level of its mRNA, may indicate the predominance of regulation of the TH synthesis at the translation level during this period. However, the content of dopamine-β-hydroxylase and its mRNA does not change during the perinatal period.

Molecular mechanisms of synthesis of noradrenaline as an inducer of development in the adrenal glands of rats in ontogenesis

The level of gene expression and the protein content of tyrosine hydroxylase and dopamine β-hydroxylase were determined. In the perinatal period of rats, when noradrenaline functions as a morphogenetic factor, the level of gene expression of these enzymes increased and the content of protein products of these genes was almost unchanged, indicating the difference in the regulatory mechanisms of their transcription and translation.

Gene expression and content of enzymes of noradrenaline synthesis in the rat organ of Zuckerkandl at the critical period of morphogenesis

Gene expression and content of the key enzymes involved in the synthesis of noradrenaline—tyrosine hydroxylase and dopamine beta-hydroxylase—was evaluated in the organ of Zuckerkandl of rats in the critical period of morphogenesis. High levels of mRNA and protein of both enzymes in the perinatal period of development and their sharp decline on day 30 of postnatal development were detected. These data indicate that the synthesis of noradrenaline in the organ of Zuckerkandl is maximum during the critical period of morphogenesis and decreases during the involution of this paraganglion.