Olga Krizanova

Calcium and ROS: A mutual interplay

Calcium is an important second messenger involved in intra- and extracellular signaling cascades and plays an essential role in cell life and death decisions. The Ca2+ signaling network works in many different ways to regulate cellular processes that function over a wide dynamic range due to the action of buffers, pumps and exchangers on the plasma membrane as well as in internal stores. Calcium signaling pathways interact with other cellular signaling systems such as reactive oxygen species (ROS). Although initially considered to be potentially detrimental byproducts of aerobic metabolism, it is now clear that ROS generated in sub-toxic levels by different intracellular systems act as signaling molecules involved in various cellular processes including growth and cell death. Increasing evidence suggests a mutual interplay between calcium and ROS signaling systems which seems to have important implications for fine tuning cellular signaling networks. However, dysfunction in either of the systems might affect the other system thus potentiating harmful effects which might contribute to the pathogenesis of various disorders.

Gene Expression of Phenylethanolamine N-Methyltransferase in Corticotropin-Releasing Hormone Knockout Mice During Stress Exposure

Summary1. 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.2. 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.3. 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.4. 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.

Cdc2/Cyclin B1 Interacts with and Modulates Inositol 1,4,5-Trisphosphate Receptor (Type 1) Functions

The resistance of inositol 1,4,5-trisphosphate receptor (IP3R)-deficient cells to multiple forms of apoptosis demonstrates the importance of IP3-gated calcium (Ca2+) release to cellular apoptosis. However, the specific upstream biochemical events leading to IP3-gated Ca2+ release during apoptosis induction are not known. We have shown previously that the cyclin-dependent kinase 1/cyclin B (cdk1/CyB or cdc2/CyB) complex phosphorylates IP3R1 in vitro and in vivo at Ser421 and Thr799. In this study, we show that: 1) the cdc2/CyB complex directly interacts with IP3R1 through Arg391, Arg441, and Arg871; 2) IP3R1 phosphorylation at Thr799 by the cdc2/CyB complex increases IP3 binding; and 3) cdc2/CyB phosphorylation increases IP3-gated Ca2+ release. Taken together, these results demonstrate that cdc2/CyB phosphorylation positively regulates IP3-gated Ca2+ signaling. In addition, identification of a CyB docking site(s) on IP3R1 demonstrates, for the first time, a direct interaction between a cell cycle component and an intracellular calcium release channel. Blocking this phosphorylation event with a specific peptide inhibitor(s) may constitute a new therapy for the treatment of several human immune disorders.

Quantitative Evaluation of Catecholamine Enzymes Gene Expression in Adrenal Medulla and Sympathetic Ganglia of Stressed Rats

Abstract: Stress‐induced changes in mRNA levels of tyrosine hydroxylase (TH), dopamine‐β‐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.

Effect of Novel Stressors on Tyrosine Hydroxylase Gene Expression in the Adrenal Medulla of Repeatedly Immobilized Rats

The activity of the sympathetic-adrenomedullary system in rats submitted to novel stressors after prior repeated or chronic stress exposure is poorly understood. The purpose of the present work was to investigate changes in adrenomedullary (AM) tyrosine hydroxylase (TH) gene expression after a single or long-term repeated exposure of rats to immobilization stress (IMMO; 42 times), as well as in repeatedly immobilized rats (41 times) exposed once to various novel heterotypic stressors. Cold exposure for 5 h, administration of insulin (INS, 5IU), or 2-deoxyglucose (2DG, 500 mg/kg) were used as novel stressors. A single exposure to cold, INS, or 2DG produced transient increases in TH mRNA levels in AM. Animals exposed to repeated homotypic IMMO stress showed permanently increased TH mRNA levels, TH activity, and protein levels; however, an exposure of such animals to heterotypic novel stressors did not induce any further changes. Thus the observed differences in TH mRNA levels in the AM of control rats and long-term repeatedly IMMO rats suggest that an adaptation to this stressor is displayed by a permanently increased TH gene expression, TH activity, and protein level. The exposure of repeatedly IMMO rats to a single episode of novel stressor does not induce exaggerated responses in TH gene expression, as some other stressors do. The mechanism of this finding could involve a central regulation and/or adrenomedullary signaling pathway(s), leading to additional modifications or accumulation of transcription factors. The precise mechanism(s) of this phenomenon remains to be elucidated.

NF-κB inhibition significantly upregulates the norepinephrine transporter system, causes apoptosis in pheochromocytoma cell lines and prevents metastasis in an animal model

Pheochromocytomas (PHEOs) and paragangliomas (PGLs) are specific types of neuroendocrine tumors that originate in the adrenal medulla or sympathetic/parasympathetic paraganglia, respectively. Although these tumors are intensively studied, a very effective treatment for metastatic PHEO or PGL has not yet been established. Preclinical evaluations of novel therapies for these tumors are very much required. Therefore, in this study we tested the effect of triptolide (TTL), a potent nuclear factor‐kappaB (NF‐κB) inhibitor, on the cell membrane norepinephrine transporter (NET) system, considered to be the gatekeeper for the radiotherapeutic agent 131I‐metaiodobenzylguanidine (131I‐MIBG). We measured changes in the mRNA and protein levels of NET and correlated them with proapoptotic factors and metastasis inhibition. The study was performed on three different stable PHEO cell lines. We found that blocking NF‐κB with TTL or capsaicin increased both NET mRNA and protein levels. Involvement of NF‐κB in the upregulation of NET was verified by mRNA silencing of this site and also by using NF‐κB antipeptide. Moreover, in vivo treatment with TTL significantly reduced metastatic burden in an animal model of metastatic PHEO. The present study for the first time shows how NF‐κB inhibitors could be successfully used in the treatment of metastatic PHEO/PGL by a significant upregulation of NET to increase the efficacy of 131I‐MIBG and by the induction of apoptosis.

Modulation of Catecholamine‐Synthesizing Enzymes in the Rat Heart by Repeated Immobilization Stress

Abstract: Stress is one of the major risk factors responsible for the increased incidence of a number of common life‐threatening disorders, predominantly of cardiovascular origin. The aim of the present study was to establish the effect of repeated immobilization stress on gene expression and protein levels of aromatic L‐amino acid decarboxylase (AADC) and phenylethanolamine N‐methyltransferase (PNMT) in cardiac left and right atria. In the process of repeated immobilization, rats were immobilized 2 h daily for 7 days and killed 3 h after the last immobilization. Gene expression was determined by regular and real‐time reverse transcription with subsequent polymerase chain reaction, and protein levels were determined by Western blot analysis. In cardiac atria, we identified mRNA for AADC and PNMT. Repeated immobilization stress did not affect AADC mRNA levels. However, repeated immobilization significantly increased PNMT mRNA levels compared with unstressed control animals. No further increase was observed compared with adapted control rats (rats immobilized six times for 2 h daily and decapitated 24 h after the sixth immobilization). AADC protein levels corresponded with mRNA levels of this enzyme. However, we were not able to detect PNMT immunoreactive protein. The observed elevation in the gene expression of PNMT mRNA levels in the heart may be involved in the increased risk of cardiovascular diseases with stress.

Quantitation of changes in gene expression of norepinephrine biosynthetic enzymes in rat stellate ganglia induced by stress.

Enzymes involved in catecholamine synthesis are present in the highest concentration in the adrenal medulla, however they were found also in other, mainly nervous tissues. The aim of our study was to quantify the exact concentration of tyrosine hydroxylase (TH) and dopamine-ss-hydroxylase (DBH) mRNA in rat stellate ganglia under control conditions and at different intervals after exposure to immobilization stress (IMO). In rats immobilized once for 2h, we determined TH and DBH mRNA in different time intervals up to 22 h after the end of the stress stimulus. TH immunoreactive protein levels were also determined in stellate ganglia. TH and DBH mRNA levels were quantified by RT-competitive-PCR. In stellate ganglia, the concentration of TH mRNA was 17+/-1.6 amol/microg of total RNA, which is approximately 30-times lower than in the adrenal medulla. The concentration of DBH mRNA in the stellate ganglia was 2601+/-203 amol/microg of total RNA, which is the concentration similar to adrenal medulla, but is 150-times higher than concentration of TH mRNA in stellate ganglia. After a single 2-h immobilization the highest elevation of TH and DBH mRNA levels was measured 22 h after the termination of the stress stimulus. Repeated immobilization (7 days, 2h daily) did not produce further increase in TH and DBH mRNA levels compared to already elevated levels in adapted control group (immobilized for 6 days, 2h daily and decapitated 22 h later). Levels of TH protein were significantly changed only after the repeated immobilization. This study compared for the first time the precise amounts of TH and DBH mRNA in rat stellate ganglia under control conditions and after immobilization stress, and indicates large differences in their concentration. TH and DBH mRNA concentrations in stellate ganglia are markedly elevated for a prolonged period of time after termination of the stress stimuli.

Localization and Regulation of Phenylethanolamine N‐Methyltransferase Gene Expression in the Heart of Rats and Mice during Stress

Abstract: Recently we have described the existence of phenylethanolamine N‐methyltransferase (PNMT) mRNA in the heart of adult rats. In this study, we report the first data on distribution of the PNMT protein in rat hearts, which follows the distribution of PNMT mRNA (high levels in the atria and low levels in ventricles). The main aim of this study was to determine the localization of the PNMT mRNA in the heart and to examine whether gene expression of this enzyme is affected by immobilization (IMO) stress in a time‐dependent manner. PNMT mRNA levels were detected in all seven studied parts of the heart (atria without and with intramural ganglion cells, ventricles, and septum), with the highest levels in the left atrium and its ganglionic part. Both Southern blot and sequencing verified the specificity of PNMT detected by RT‐PCR. Single IMO for 2‐h increased gene expression of PNMT, as determined by both RT‐PCR and Real‐Time PCR in the right and left atria. Surprisingly, the ganglionic parts of the atria did not respond to stress stimulation. Peak levels of PNMT mRNA were found in the 3‐h interval after the IMO terminated, and also 24 h after the first or sixth IMO. Expression of aromatic L‐amino acids decarboxylase and dopamine‐beta‐hydroxylase has also been detected in the heart of control and stressed rats. In the atria, the effect of stress is clearly modulated by glucocorticoids, since in mice with corticotrophin‐releasing hormone knocked out gene the immobilization‐induced increase in the PNMT mRNA levels seen in wild‐type animals was abolished. Thus, our data have shown that gene expression of the PNMT is localized, not predominantly in cardiac ganglion cells, but in a wide range in atrial cardiomyocytes. Mechanism responsible for the regulation of stress‐induced increase of PNMT gene expression in cardiac atria is clearly dependent on the presence of glucocorticoids.

Immobilization stress elevates IP3 receptor mRNA in adult rat hearts in a glucocorticoid-dependent manner

Gene expression of the type 1 and 2 inositol 1,4,5‐trisphosphate (IP3) receptors in the rat cardiac atria and ventricles and their possible modulation by single immobilization stress was studied. Single immobilization stress significantly elevated mRNA levels for both types of these receptors. To evaluate the involvement of glucocorticoids in the modulation of the gene expression of IP3 receptors by immobilization stress, we used adrenalectomized and/or hypophysectomized rats. Since adrenalectomy and/or hypophysectomy completely abolished increase in IP3 receptors mRNA levels after the immobilization, we conclude that immobilization stress elevates mRNA of type 1 and 2 IP3 receptors, mainly through the glucocorticoid responsive element.