Uranyl acetate modulates gene expression and protein levels of the type 2, but not type 1 inositol 1,4,5-trisphosphate receptors in mouse kidney.

Nephrotoxic effect of uranium is already well documented. Nevertheless, little is known about the effect of uranium on calcium homeostasis and calcium transport systems. Calcium released from endoplasmic reticulum through special calcium release channels--inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs)--serves as a main source of cytosolic calcium signaling in the majority of cell types. To contribute to understanding mechanism of toxicity of the uranyl acetate (UA), we focused on modulation of the gene expression, protein levels and activity of IP3 receptor's intracellular calcium channels by UA in mouse kidney. We have found that UA did not affect mRNA and protein levels of the type 1 IP3Rs, but increased mRNA and also protein levels of the type 2 IP3 receptors in kidney. Nevertheless, IP3-induced calcium release was decreased by addition of UA. We assume that decreased activity of IP3 receptors due to the acute exposure to UA results in feedback, which triggers activation of IP3R2 expression. Thus, inhibition of calcium release and increased levels of the type 2 IP3 receptors might participate, at least partially, in UA-induced nephrotoxicity.

Adrenergic and calcium modulation of the heart in stress: from molecular biology to function.

There is strong evidence about the importance of catecholamines and calcium signaling in heart function. Also, interaction of these two systems is well documented. Catecholamines signal through adrenergic receptors, and further activate calcium transport either from the extracellular space, or from the intracellular calcium stores. This review summarizes current knowledge on catecholamine production in the heart, with special focus on the final enzyme in the catecholamine synthesizing pathway, phenylethanolamine N-methyltransferase (PNMT), in different cell types in the heart. Further, signaling through different types of adrenergic receptors in physiological conditions and after exposure to different stressors is discussed. Also, part of this review considers activation of an intracellular calcium transport system via inositol 1,4,5-trisphosphate receptor and to possible functional consequences in control and stress conditions.

Modulation of expression of Na+/Ca2+ exchanger in heart of rat and mouse under stress.

AIM: The Na(+)/Ca(2+) exchanger (NCX) is a major Ca(2+) extrusion system in the plasma membrane of cardiomyocytes and an important component participating on the excitation-contraction coupling process in muscle cells. NCX1 isoform is the most abundant in the heart and is known to be changed after development of ischaemia or myocardial infarction. Objective of this study was to investigate the effect of stress factors (immobilization, cold and short-term hypoxia) on the expression of NCX1, in vivo, in the heart of rat and mouse. METHODS: We compared gene expression and protein levels of control and stressed animals. The activity of NCX was measured by the whole cell configuration using the patch clamp. We also measured physiological parameters of the heart in physiological conditions and under ischaemia-reperfusion to compare response of control and stressed hearts. RESULTS: We have found that only strong stress stimulus (hypoxia, immobilization) applied repeatedly for several days elevated the NCX1 mRNA level. Cold, which is a weaker stressor that activates mainly sympathoneural, and only marginally adrenomedullary system did not affect the gene expression of NCX1. Thus, from these results it appears that hormones produced by the adrenal medulla (mainly adrenaline) might be involved in this process. To study possible mechanism of the NCX1 regulation by stress, we focused on the possible role of the hypothalamo-pituitary-adrenocortical pathway in the activation of catecholamine synthesis in the adrenal medulla. We have already published that cortisol affects activity, but not the gene expression of NCX1. In this work, we used corticotropin-releasing hormone (CRH) knockout mice, where secretion of corticosterone and subsequently adrenaline is significantly suppressed. As no increase in NCX1 mRNA was observed in CRH knockout mice due to immobilization stress, we proposed that adrenaline (probably regulated via corticosterone) is involved in the regulation of NCX1 gene expression during stress. CONCLUSIONS: The gene expression and protein levels of the NCX1 are increased by the strong stress stimuli, e.g. hypoxia, or immobilization stress. The activity of NCX1 is decreased. Based on these results, we assume that the gene expression of NCX is increased as a consequence of suppressed activity of this transport system.

Hypoxia modulates gene expression of IP3 receptors in rodent cerebellum.

Hypoxic brain cell injury is a complex process that results from a series of intracellular events. In this work, we tested whether severe hypoxia for 6 h can affect gene expression and protein levels of intracellular calcium channels, ryanodine receptors, and inositol 1,4,5-trisphosphate receptors in mouse cerebellum. In addition, we tested the effect of hypoxia on cerebellar granular cells of rats. We have found that gene expression of types 1 and 2 IP(3) receptors is significantly increased after the exposure of mice to hypoxic stimulus for 6 h and also in rat cerebellar granular cells. Increased gene expression of IP(3) receptors was reflected in increased protein levels of these channels as well. In this process, reactive oxygen species are most probably involved, as antioxidant quercetin abolished hypoxia-induced increase in both types 1 and 2 IP3 receptor. Ryanodine receptors of types 1 and 2 and sarco(endo)plasmic reticulum Ca(2+)-ATPase were not affected by hypoxia on the level of messenger RNA. To test physiological consequences, we measured levels of intracellular calcium. We observed significantly elevated calcium level in hypoxic compared to normoxic cells. Deeper understanding of mechanisms, through which hypoxia regulates intracellular calcium, could point towards the development of new therapeutic approaches to reduce or suppress the pathological effects of cellular hypoxia, such as those seen in stroke or ischemia.

Gene expression of phenylethanolamine N-methyltransferase in corticotropin-releasing hormone knockout mice during stress exposure.

AIMS: Epinephrine (EPI) synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) is primarily localized in the adrenal medulla (AM). We have recently described existence of the PNMT gene expression in cardiac atria and ventricles and in sympathetic ganglia of adult rats and mice. The aim of the present work was to study regulation of the PNMT gene expression in corticotropin-releasing hormone knockout mice (CRH KO) and matched control wild-type mice (WT) under normal and stress conditions. METHODS: Levels of the PNMT mRNA were determined by RT-PCR; PNMT immunoprotein and protein of transcription factor EGR-1 by Western Blot. Plasma EPI and corticosterone (CORT) levels were determined by radioenzymatic and RIA methods. Immobilization (IMMO) was used as a stressor. RESULTS: Stress-induced increases in the PNMT mRNA and protein levels observed in WT mice were almost completely absent in CRH KO mouse adrenal medulla, stellate ganglia, and cardiac atria, while ventricular PNMT mRNA elevation was not CRH-dependent. Plasma EPI and CORT levels were markedly reduced in CRH KO compared to WT mice both before and after the stress. Levels of EGR-1, crucial transcription factor for regulation of the PNMT were highly increased in stressed WT and CRH KO mice in cardiac areas, but not in the adrenal medulla. CONCLUSIONS: Data show that the CRH deficiency can markedly prevent immobilization-triggered induction of the PNMT mRNA and protein levels in the adrenal medulla and stellate ganglia. Reduced plasma epinephrine and corticosterone levels and adrenal medullary EGR-1 protein levels in CRH knockout versus WT mice during stress indicate that the HPA axis plays a crucial role in regulation of the PNMT gene expression in these organs. Cardiac atrial PNMT gene expression with stress is also dependent on intact HPA axis. However, in cardiac ventricles, especially after the single stress exposure, its expression is not impaired by CRH deficiency. Since cardiac EGR-1 protein levels in CRH KO mice are also not affected by the single stress exposure, we propose existence of different regulation of the PNMT gene expression, especially in the cardiac ventricles.Overall, our findings reveal that the PNMT gene expression is regulated through the HPA in both sympathoadrenal system and the heart and also via EGR-1 in the adrenal medulla, but apparently not in the heart. Regulation of the PNMT gene expression in various compartments of heart includes both corticosterone-dependent and independent mechanisms.

Identification of phenylethanolamine N-methyltransferase gene expression in stellate ganglia and its modulation by stress.

Phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) is the terminal enzyme of the catecholaminergic pathway converting noradrenaline to adrenaline. Although preferentially localized in adrenal medulla, evidence exists that PNMT activity and gene expression are also present in the rat heart, kidney, spleen, lung, skeletal muscle, thymus, retina and different parts of the brain. However, data concerning PNMT gene expression in sympathetic ganglia are still missing. In this study, our effort was focused on identification of PNMT mRNA and/or protein in stellate ganglia and, if present, testing the effect of stress on PNMT mRNA and protein levels in this type of ganglia. We identified both PNMT mRNA and protein in stellate ganglia of rats and mice, although in much smaller amounts compared with adrenal medulla. PNMT gene expression and protein levels were also increased after repeated stress exposure in stellate ganglia of rats and wild-type mice. Similarly to adrenal medulla, the immobilization-induced increase was probably regulated by glucocorticoids, as determined indirectly using corticotropin-releasing hormone knockout mice, where immobilization-induced increase of PNMT mRNA was suppressed. Thus, glucocorticoids might play an important role in regulation of PNMT gene expression in stellate ganglia under stress conditions.

Gene expression of the phenylethanolamine N-methyltransferase is differently modulated in cardiac atria and ventricles.

Phenylethanolamine N-methyltransferase (PNMT) is a final enzyme in catecholamine synthesizing cascade that converts noradrenaline to adrenaline. Although most profuse in adrenal medulla, PNMT is expressed also in the heart, particularly in cardiac atria and ventricles. In atria, the PNMT mRNA is much more abundant compared to ventricles. In present study we aimed to find out whether there is a difference in modulation of the PNMT gene expression in cardiac atria and ventricles. We used three methodological approaches: cold as a model of mild stress, hypoxia as a model of cardiac ischemic injury, and transgenic rats (TGR) with incorporated mouse renin gene (mREN-2)27, to determine involvement of renin-angiotensin pathway in the PNMT gene expression. We have found that PNMT gene expression was modulated differently in cardiac atria and ventricles. In atria, PNMT mRNA levels were increased by hypoxia, while cold stress decreased PNMT mRNA levels. In ventricles, no significant changes were observed by cold or hypoxia. On the other hand, angiotensin II elevated PNMT gene expression in ventricles, but not in atria. These results suggest that PNMT gene expression is modulated differently in cardiac atria and ventricles and might result in different physiological consequences.

Inhibitory effect of formalin administration on immobilization-induced epinephrine release.

Injection of formalin is used as a classical painful stressor that produces a biphasic nociceptive response consisting of a 1- to 10-min early phase and a later phase 30 to 240 min after injection. The period between these two phases, called "interphase," is characterized by attenuated nociception. We evaluated the response of catecholamine plasma levels to formalin-induced pain stress with special attention to these three time periods. Subcutaneous injection of 4% formalin (0.2 mL/100 g bw) into the hind limb produced a slight reduction of plasma epinephrine levels in the first 15 min, which was followed by a significant increase that remained high up to 120 min after injection. Norepinephrine levels increased immediately after injections and remained high from 30 until 120 min. To test the effect of formalin injection in a stressful condition, we exposed animals to 2 h immobilization stress. In the first experiment, formalin was injected before the start of immobilization. A significant decrease of plasma epinephrine levels was measured up to 25 min post-injection, whereas plasma norepinephrine levels remained high. A second formalin injection during immobilization was as effective as the first one: It depleted plasma epinephrine levels from 5 to 15 min post-injection without significant changes in norepinephrine levels. In the second experiment, formalin given after the beginning of immobilization produced a significant decrease of epinephrine levels 15 min after the injection and produced a significant increase 60 min after injection. The plasma norepinephrine levels were significantly increased by 40 min post-injection. The data show that the inhibitory process during the interphase of formalin test is able to significantly decrease epinephrine release not only during basal conditions but also during exposure to a severe stressor, such as immobilization without suppression of plasma norepinephrine levels.

Pivotal role of an endogenous tetrahydroisoquinoline, salsolinol, in stress- and suckling-induced release of prolactin.

In mammals, the role of a prolactin-releasing factor (PRF) in the acute changes of prolactin (PRL) secretion that usually occur after challenges (e.g., suckling stimulus or stress) of homeostasis has been suspected for a long time. We have recently observed that 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, salsolinol (SAL), produced by the hypothalamus and the neuro-intermediate lobe (NIL) of the pituitary gland, can selectively release PRL from the anterior lobe (AL). Moreover, binding sites for SAL have been detected in areas like median eminence, NIL, and AL. It has been proposed that SAL is a putative endogenous PRF. We have also found that a structural analogue of SAL, 1-methyl-3,4-dihydroisoquinoline (1MeDIQ), is able to block dose-dependently SAL-, suckling-, and immobilization (IMO) stress-induced release of PRL without having any influence on alpha-methyl-p-tyrosine (alphaMpT)-induced PRL responses. Neither SAL nor 1MeDIQ has any effect on alpha-melanocyte-stimulating hormone (alphaMSH), adrenocorticotrophic hormone (ACTH), beta-endorphin (beta-END) and arginine-vasopressin (AVP) secretion. Moreover, SAL-induced PRL response was attenuated in male rats pretreated with dexamethasone (DEX). These results strongly suggest that SAL has an important role in the regulation of PRL release induced by physiologic and environmental stimuli; therefore, it can be considered as the strongest candidate for being the PRF in the hypothalamo-hypophysial system. Our findings also indicate that the adrenal steroids may play an inhibitory feedback role in SAL-mediated PRL response.

Effect of immobilization on in vitro thyrotropin-releasing hormone release from brain septum in wild-type and corticotropin-releasing hormone knock-out mice.

There is considerable evidence linking alcohol consumption, sedation, and thyrotropin-releasing hormone (TRH) in the brain septum. We have shown that ethanol in clinically relevant concentrations can in vitro induce TRH release from the septum by a mechanism involving neuronal swelling. Corticotropin-releasing hormone-deficient (CRH-KO) mice serve as an interesting model to help us understand the role of CRH in the regulation of different neuroendocrine systems. The aim of this study was to compare TRH release activity in the brain septum at basal and stress conditions in CRH-KO mice and their wild-type (WT) littermates. Experimental mice were decapitated immediately or 3 h after single (2 h) or repeated (seven times for 2 h daily) immobilization stress. The brain septum was immediately cut out and incubated to measure basal-, ethanol-, and hyposmosis-stimulated TRH release in vitro. Ethanol in isosmotic medium or hyposmotic medium stimulated TRH release from mice septal explants from WT and CRH-KO mice. The response was disturbed immediately after immobilization and recovered 3 h later. Our results show that immobilization stress transiently affects the TRH system in brain septum. Inborn absence of CRH does not affect septal TRH and its response to ethanol before and 3 h after immobilization.

Quantitative evaluation of catecholamine enzymes gene expression in adrenal medulla and sympathetic Ganglia of stressed rats.

Stress-induced changes in mRNA levels of tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT) have been expressed as relative arbitrary units compared with a control group. The aim of this study was to quantify basal and stress-induced levels of TH, DBH, and PNMT mRNAs in rat adrenal medulla (AM) and stellate ganglia (SG) by the RT-competitive PCR method using corresponding competitors of known concentration. In rats stressed by immobilization (IMO) once for 2 h, the concentration of mRNAs was determined in various intervals after the end of stress stimulus. In SG, the basal concentration of TH mRNA was 0.017 amol/ng of total RNA, which is approximately 30 times lower than in the AM (0.460 amol/ng RNA). The basal concentration of DBH mRNA in SG was 2.60 amol/ng of total RNA, which is about 150 times more than TH mRNA in SG but only two times less than DBH mRNA in the AM in which PNMT mRNA is present in the highest concentration. After a single 2-h IMO, the peak elevation of TH and DBH mRNA concentration in SG occurred 24 h after the termination of stress stimulus, when their AM mRNA concentrations were already at control values. Presence of PNMT mRNA levels in the SG, of control and stressed rats has been demonstrated for the first time. Repeated IMO (7 days, 2 h daily) did not produce further increase in the mRNA concentrations compared with the elevated values found in adapted control groups. Levels of TH protein were significantly increased only after repeated IMO in SG and AM. Thus, our data show for the first time the exact concentrations of TH, DBH, and PNMT mRNA in SG and AM of rats under control and stress conditions. The lowest concentration of TH mRNA in the AM and SG supports the hypothesis that tyrosine hydroxylation is the rate-limiting step in catecholamine biosynthesis.

Catecholamine synthesizing enzymes and their modulation by immobilization stress in knockout mice.

The c-fos knockout mice (c-fos KO) and corticotropin-releasing hormone knockout mice (CRH KO) can serve as interesting models for studying mechanisms involved in response of the organism to stress, focused mainly on the hypothalamic-pituitary-adrenal (HPA) axis and sympathoadrenal system (SAS). The present study focused on the investigation of changes in gene expression of catecholamine biosynthesizing enzymes tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT) in adrenal medulla of c-fos KO and CRH KO mice stressed by immobilization. Levels of TH, DBH, and PNMT mRNA were determined by reverse transcription-polymerase chain reaction (RT-PCR). Single immobilization for 2 h significantly increased adrenomedullary TH, DBH, and PNMT mRNA levels in both c-fos KO and wild-type (WT) mice compared to unstressed controls. In CRH KO mice, PNMT gene expression was not increased to the same extent after single, but especially after repeated immobilization as in WT mice, in contrast to TH and DBH mRNA levels. Thus, our data indicate that CRH deficiency can influence the PNMT mRNA level in adrenal medulla during stress, confirming the idea that the HPA axis plays the crucial role in PNMT gene regulation in mice. On the other hand, c-Fos protein probably does not play a crucial role in TH, DBH, and PNMT gene expression in adrenal medulla under stress conditions.

Identification of the aromatic L-amino acid decarboxylase gene expression in various mice tissues and its modulation by immobilization stress in stellate ganglia.

Despite of the fact that the impact of various stressful stimuli on catecholamine biosynthetic enzyme gene expression, activity and immunoreactive protein has been intensively studied, less is known about the aromatic L-amino acid decarboxylase (AADC), the enzyme, which catalyzes decarboxylation of L-dihydroxyphenylalanine to dopamine. We focused on the identification of AADC mRNA and immunoprotein in various mice tissues and detected both in selected mice neuronal tissues (adrenal medulla, sympathetic stellate and cervical ganglia) and also in non-neuronal tissues (liver, spleen, kidney and all four parts of the heart). Surprisingly, although we failed to detect AADC mRNA in mice thymus, lungs and abdominal fat, we found presence of the AADC immunoprotein in lungs as well as in the abdominal fat. We also tested the hypothesis, whether single or repeated immobilization stress can affect the AADC mRNA or immunoprotein levels in mice stellate ganglia. We revealed that single immobilization stress exposure did not affect the AADC mRNA or immunoprotein levels, while repeated immobilization stress produced significant elevation of both, AADC mRNA and immunoprotein levels in stellate ganglia. The aromatic L-amino acid decarboxylase is generally not considered to be limiting in regulation of the catecholamine biosynthesis. However, our data suggest a possible participation of this enzyme in the regulation of catecholamine biosynthesis in stellate ganglia of repeatedly stressed mice.

Stress- as well as suckling-induced prolactin release is blocked by a structural analogue of the putative hypophysiotrophic prolactin-releasing factor, salsolinol.

Prolactin is secreted from the anterior lobe of the pituitary gland in response both to suckling and to stress. We recently observed that 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol), produced in the neurointermediate lobe of the pituitary gland, as well as in the medial basal hypothalamus, can selectively release prolactin from the anterior pituitary. Therefore, it has been proposed that salsolinol is a putative endogenous prolactin-releasing factor (PRF). Here, we report that one structural analogue of salsolinol, 1-methyl-3,4-dihydroisoquinoline (1MeDIQ), can block salsolinol-induced release of prolactin, but does not affect prolactin release in response to thyrotropin releasing hormone (TRH), alpha-methyl-p-tyrosine (alpha MpT) (an inhibitor of tyrosine hydroxylase), domperidone (a D(2) dopamine receptor antagonist), or 5-hydroxytryptophan (5-HTP), a precursor of serotonin). 1MeDIQ profoundly inhibited suckling-, immobilization-, as well as formalin-stress induced prolactin release without any influence on corticosterone secretion. The 1MeDIQ-induced reduction in prolactin response to immobilization stress was dose-dependent. These results suggest that salsolinol can play a pivotal role in the regulation of prolactin release induced by either physiological (suckling) or environmental (stress) stimuli.

[Use of knock-out mice in studies of stress reactions]. / Vyuzitie "knock-out" mysí pri stúdiu stresovej reakcie organizmu.

Modern human population intimately recognizes the stress reaction of the organism, which has developed as a response to variety physical and/or psychical stressors. This reaction represents not only a complex of adaptive mechanisms enabling the successful overcoming of the conflicting situation, but in its pathological form it can significantly contribute to the development of serious diseases, from carcinogenic, cardiovascular, gastrointestinal, up to the psychical. New, developing scientific disciplines permit to widen knowledge of the basis of this undesirable side of the stress. Transgenic animals, which either has integrated the gene of interest, or studied gene is not functioning (i.e. knockout animals), permit at least partially to study the physiological importance of the product of this gene in the organism. In our review we described in detail three lines of knockout mice--with the knockout gene for the dopamine-beta-hydroxylase, corticoliberin and c-fos--that significantly contribute to the regulation of the stress response.

Stress increases gene expression of phenylethanolamine N-methyltransferase in spleen of rats via pituitary-adrenocortical mechanism.

Gene expression of phenylethanolamine N-methyltransferase (PNMT), the enzyme catalyzing conversion of norepinephrine to epinephrine, has been detected in rat spleen using the reverse transcription polymerase chain reaction. PNMT identity was subsequently verified by Southern blots. Localization of the spleen cells responsible for the PNMT gene expression was investigated by the in situ hybridization and PNMT mRNA was found to be present in the white pulp. The hypothesis that stress may produce an increase in PNMT gene expression in rat spleen was tested and a robust rise in the relative abundance of PNMT mRNA levels was observed after a single or repeated immobilization (about 80%). Adrenalectomy or hypophysectomy completely prevented the immobilization-induced increase in spleen PNMT mRNA levels, suggesting that stress-induced PNMT gene expression in the spleen is regulated predominantly via pituitary-adrenocortical axis. In control animals, however, spleen PNMT was not significantly affected by the ectomies and therefore basal PNMT gene expression might be regulated by different mechanism(s).Thus, PNMT gene expression in the rat spleen is exaggerated by stress stimuli, suggesting its role in physiological regulations.

Identification of tyrosine hydroxylase gene expression in rat spleen.

This study was aimed to identify tyrosine hydroxylase (TH) gene expression in the rat spleen under basal and stress conditions. Using the reverse transcription polymerase chain reaction we did not detect TH mRNA in rat spleen either in control, or immobilized animals. Semi-nested PCR revealed a clear signal, demonstrating that TH mRNA is formed in the spleen, although in low abundance. We also detected both, TH immunoreactive protein and TH activity in the rat spleen that were in higher abundance than expected from the mRNA levels. This study identifies, for the first time, TH gene expression in rat spleen. Since TH protein and activity are present in the spleen in much higher abundance compared to corresponding mRNA, the majority of TH protein is most probably supplied by the sympathetic innervation of spleen.

Hypokalemia-induced ultrastructural, histochemical and connexin-43 alterations resulting in atrial and ventricular fibrillations.

Perfusion of the isolated guinea pig heart with hypokalemic solution provide simple model for examination of the molecular mechanisms involved in the incidence of atrial and/or ventricular fibrillations. The results point out that dispersion of the metabolic and subcellular alterations and heterogenously impaired intercellular coupling might account for electrical disturbances and desynchronization of the myocardium thus facilitate occurrence of fibrillation.