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We explored possibility that sodium/calcium exchanger 1 (NCX1) is involved in pH modulation and apoptosis induction in GYY4137 treated cells. We have shown that although 10 days treatment with GYY4137 did not significantly decreased volume of tumors induced by colorectal cancer DLD1 cells in nude mice, it already induced apoptosis in these tumors. Treatment of DLD1 and ovarian cancer A2780 cells with GYY4137 resulted in intracellular acidification in a concentration-dependent manner. We observed increased mRNA and protein expression of both, NCX1 and sodium/hydrogen exchanger 1 (NHE1) in DLD1-induced tumors from GYY4137-treated mice. NCX1 was coupled with NHE1 in A2780 and DLD1 cells and this complex partially disintegrated after GYY4137 treatment. We proposed that intracellular acidification is due to uncoupling of NCX1/NHE1 complex rather than blocking of the reverse mode of NCX1, probably due to internalization of NHE1. Results might contribute to understanding molecular mechanism of H2S–induced apoptosis in tumor cells.

Although paclitaxel (PTX) is potent chemotherapeutic agent commonly used in variety of cancers, in colorectal carcinoma its usage is excluded because of low effectivity. Up to now, some experimental attempts were utilized to improve sensitivity of colorectal carcinoma to PTX. We used a slow sulfide donor GYY4137 to increase sensitivity of colorectal carcinoma cells to PTX. As a model of colorectal carcinoma, we utilized three different cell lines - HCT116, SW620 and DLD1. We compared IC50 for PTX and PTX/GYY4137, cell cycle, apoptosis, ATP levels and changes in intracellular pH. We observed significant decrease in IC50 levels in PTX/GYY4137 groups compared to PTX in all three cell lines. PTX arrested cell cycle in G2/M phase. Differences in S phase were observed in HCT116 and DLD1 cells treated with 20 nM PTX/GYY4137, but not in SW620 cell. GYY4137 increased early, but not late phase of apoptosis. This increase was not detected in non-cancer EAHy926 cells. Upregulation of IP

Exogenous and endogenously produced sulfide derivatives, such as H2S/HS-/S2-, polysulfides and products of the H2S/S-nitrosoglutathione interaction (S/GSNO), affect numerous biological processes in which superoxide anion (O2-) and hydroxyl (OH) radicals play an important role. Their cytoprotective-antioxidant and contrasting pro-oxidant-toxic effects have been reported. Therefore, the aim of our work was to contribute to resolving this apparent inconsistency by studying sulfide derivatives/free radical interactions and their consequent biological effects compared to the antioxidants glutathione (GSH) and Trolox. Using the electron paramagnetic resonance (EPR) spin trapping technique and O2-, we found that a polysulfide (Na2S4) and S/GSNO were potent scavengers of O2- and cPTIO radicals compared to H2S (Na2S), GSH and Trolox, and S/GSNO scavenged the DEPMPO-OH radical. As detected by the EPR spectra of DEPMPO-OH, the formation of OH in physiological solution by S/GSNO was suggested. All the studied sulfide derivatives, but not Trolox or GSH, had a bell-shaped potency to decompose H2O2 and produced OH in the following order: S/GSNO > Na2S4 ≥ Na2S > GSH = Trolox = 0, but they scavenged OH at higher concentrations. In studies of the biological consequences of these sulfide derivatives/H2O2 properties, we found the following: (i) S/GSNO alone and all sulfide derivatives in the presence of H2O2 cleaved plasmid DNA; (ii) S/GSNO interfered with viral replication and consequently decreased the infectivity of viruses; (iii) the sulfide derivatives induced apoptosis in A2780 cells but inhibited apoptosis induced by H2O2; and (iv) Na2S4 modulated intracellular calcium in A87MG cells, which depended on the order of Na2S4/H2O2 application. We suggest that the apparent inconsistency of the cytoprotective-antioxidant and contrasting pro-oxidant-toxic biological effects of sulfide derivatives results from their time- and concentration-dependent radical production/scavenging properties and their interactions with O2-, OH and H2O2. The results imply a direct involvement of sulfide derivatives in O2- and H2O2/OH free radical pathways modulating antioxidant/toxic biological processes.

Cellular differentiation is the process, by which a cell changes from one cell type to another, preferentially to the more specialized one. Calcium fluxes play an important role in this action. Differentiated NG108-15 or PC12 cells serve as models for studying neuronal pathways. NG108-15 cell line is a reliable model of cholinergic neuronal cells. These cells differentiate to a neuronal phenotype due to the dibutyryl cAMP (dbcAMP) treatment. We have shown that a slow sulfide donor - GYY4137 - can also act as a differentiating factor in NG108-15 cell line. Calcium is an unavoidable ion required in NG108-15 cell differentiation by both, dbcAMP and GYY4137, since cultivation in EGTA completely prevented differentiation of these cells. In this work we focused primarily on the role of reticular calcium in the process of NG108-15 cell differentiation. We have found that dbcAMP and also GYY4137 decreased reticular calcium concentration by different mechanisms. GYY4137 caused a rapid decrease in type 2 sarco/endoplasmic calcium ATPase (SERCA2) mRNA and protein, which results in lower calcium levels in the endoplasmic reticulum compared to the control, untreated group. The dbcAMP revealed rapid increase in expression of the type 3 IP3 receptor, which participates in a calcium clearance from the endoplasmic reticulum. These results point to the important role of reticular calcium in a NG108-15 cell differentiation.

Objectives Stress is accompanied also by a rise in splenic catecholamines (CAs). However, indications about endogenous CA production in the spleen exist but there are no data about the cellular source of this production and possible modification by stress. Therefore, our aim was to investigate whether splenic T- and B-cells are one of main sources in the spleen expressing tyrosine hydroxylase (TH), enzyme crucial for CA biosynthesis, and phenylethanolamine N-methyltransferase (PNMT) which is necessary for epinephrine production. We also investigated whether stress is able to modify expression of both enzymes and CA levels within these cell fractions as well as tried to explain functional consequences of changes observed. Results T-cells contain higher levels of TH mRNA than B-cells although protein levels appeared similar. On contrary, the PNMT mRNA and protein were higher in B-cells, which appeared to be the main source of PNMT in the spleen. T-cells increased TH and PNMT expression after acute stress while similar rise was observed in B-cells after repeated stress, most probably as a consequence of higher CA turnover in both cell populations. The rise in TH and PNMT was accompanied by an elevation of Bax/Bcl-2 mRNA ratio, number of apoptotic cells and also by a decline of IFN-γ mRNA in both cell types. Reduction of IL-2 and IL-4 mRNA was also observed in B-cells. Conclusion Stress-induced stimulation of endogenous CA biosynthesis in lymphocytes is dependent on the type of lymphocyte population and duration of stressor and leads to attenuated IFN-γ expression and induction of apoptosis. These changes might contribute to dysregulation of specific immune functions involving T- and B-cells and may decrease the ability to cope with intracellular agents following stress situations.

Although c-Fos protein is one of the principal molecules in intracellular signaling, c-fos gene disruption is associated with alterations in neuronal functions that do not correspond to its importance in function. The aim of the study was to evaluate the changes of dopaminergic system together with acetylcholinesterase (AChE) in c-fos disruption (KO). KO male mice showed an increase in D₁-like receptor (279% of WT) and D₂-like receptor (345% of WT) binding sites in the cortex. On the gene expression level (assessed by real-time PCR), lower quantities of D₁R-mRNA (0.64) and D₅R-mRNA (0.6) were found in females when compared to males in the frontal cortex, higher D₂R-mRNA in the parietal (1.43) and temporal (2.64) cortex and lower AChE-mRNA (0.67). On the contrary, female striatum contained higher level of D₂R-mRNA (1.62) and AChE-mRNA (1.57) but lower level of D₃R-mRNA (0.73). Hypothalamic D₁R-mRNA, D₂R-mRNA and D₄R-mRNA were higher in females (1.38, 1.63, and 1.68, respectively). Disruption of c-fos increased selectively D₅R-mRNA (1.31) in male parietal cortex, D₂R-mRNA (1.72) in male temporal cortex, and cerebellar D₂R-mRNA in both males (1.43) and females (1.42), respectively. In females, we found rather decrease in DR-mRNA. Multiple correlations in mRNA quantities (in WT mice) were found, which changed considerably upon c-fos KO. Main interactions in WT were inter-regional, CNS of KO underwent an extensive restructuring comprising intraregional interactions in the frontal cortex, hypothalamus, and cerebellum. These changes in DR (between others) could be considered as one of the adaptive mechanisms in c-fos KO mice.

The present study addresses the hypothesis that adrenergic regulation modulates the effect of apoptosis. Therefore we studied, whether α1-adrenergic receptor's agonist phenylephrine (PE) can affect or induce apoptosis in rat pheochromocytoma (PC12) cells. We have shown that PE treatment did not increase level of the apoptosis, or level of the caspase 3 mRNA. When apoptosis was induced in the presence of PE, caspase 3 mRNA was significantly increased, while the percentage of apoptotic cells remained unchanged compared to apoptotic group without PE. During this process, α1D-, β2- and β3-adrenergic receptors (ARs) were upregulated. Since all these three types of ARs are differently localized in the cell, we assume that mutual communication of all three ARs is crucial to participate in this signaling and during development of apoptosis, some of these systems might translocate. Another important system in handling noradrenaline during apoptosis might be noradrenaline transporter (NET), since it was downregulated in apoptotic cells treated with PE, compared to untreated apoptotic cells. However, precise mechanism of mutual communication among all these systems remains to be elucidated.

The aim of this work was to characterize the effect of bongkrekic acid (BKA), atractyloside (ATR) and carboxyatractyloside (CAT) on single channel properties of chloride channels from mitochondria. Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and single chloride channel currents were measured in 250/50 mM KCl cis/trans solutions. BKA (1-100 microM), ATR and CAT (5-100 microM) inhibited the chloride channels in dose-dependent manner. The inhibitory effect of the BKA, ATR and CAT was pronounced from the trans side of a BLM and it increased with time and at negative voltages (trans-cis). These compounds did not influence the single channel amplitude, but decreased open dwell time of channels. The inhibitory effect of BKA, ATR and CAT on the mitochondrial chloride channel may help to explain some of their cellular and/or subcellular effects.

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., Graphical abstract, Highlights • Calcium and ROS act as signaling molecules inside the cell and their pathways can interact. • The mutual interplay of calcium and ROS is required for the fine tuning of signaling. • Failure in the interplay results in dysfunction and pathologies.

Previously we have shown that inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are abundantly expressed in the atria of rat hearts. Since arrangement of atria is very heterogeneous, in this work we focused on the precise localization of IP3 receptors in the left atrium, where the gene expression of the type 1 IP3R was the highest. The mRNA levels of the IP3 type 1 receptors in the left atrium, left ventricle and myocytes were determined using real-time polymerase chain reaction and Taqman probe. For precise localization, immunohistochemistry with the antibody against type 1 IP3Rs was performed. The mRNA of type 1 IP3 receptor was more than three times higher in the left atrium than in the left ventricle, as determined by real-time PCR. Expression of the type 1 IP3 receptor mRNA was higher in the atria, especially in parts containing cardiac ganglion cells. The atrial auricles, which are particularly free of ganglion cells, and the ventricles (wall of the right and left ventricle and ventricular septum) contained four to five times less IP3 receptors than atrial samples with ganglia. IP3R type 1 immunoreactivity detected by a confocal microscope attributed the most condensed signal on ganglionic cells, although light immunoreactivity was also seen in cardiomyocytes. These results show that type 1IP3 receptors predominate in intrinsic neuronal ganglia of cardiac atria.

Inositol 1,4,5-trisphosphate (IP3) receptors are calcium-releasing channels localized on the sarcoplasmic reticulum. IP3 receptors mediate the calcium mobilizing effect of a wide range of hormones, cytokines, and neurotransmitters and play an important role in variety of cell functions. The aim of this work was to study, how partial depletion of catecholamines affects the gene expression and protein levels of the type 1 IP3 receptors in rat heart. The type 1 IP3 receptor mRNA levels were studied in the left cardiac atrium and ventricle of rats treated with 6-hydroxydopamine (6-OHDA) in control and stressed conditions. The 6-OHDA produces anatomical and functional denervation resulting in decreased levels of noradrenaline and adrenaline. We also used corticoliberin (CRH) knockout mice, where secretion of adrenaline is significantly suppressed. Administration of 6-OHDA significantly decreases mRNA levels of the type 1 IP3 receptor in both, the left atrium and the left ventricle, while the gene expression of the sarcoplasmic reticular Ca2+-ATPase (SERCA 2) was unaffected. CRH knockout mice possess markedly lower levels of the type 1 IP3 receptor mRNA compared to wild-type mice in both, control and stressed conditions. These data point to the adrenergic modulation of the type 1 IP3 receptors in the rat hearts.

Inositol 1,4,5-trisphosphate (IP(3)) receptors belong to the intracellular calcium channels that release calcium from the intracellular stores after binding IP(3). Three types of IP(3) receptors occurred in a tissue specific manner and different promoters direct their gene expression. Thus, understanding of the transcriptional regulation is the first step towards comprehension of the function of these receptors. Since the retinoic acid activates RARE and AP2 transcription factors, the present study focuses on determination of whether or not expression of type 1 and 2 IP(3) receptors is modulated by retinoic acid in selected brain areas. We have found that mRNA levels of the type 1 IP(3) receptors were decreased significantly in cerebellum and hypothalamus, but not in the brain stem of rats treated with retinoic acid, compared to untreated littermates. The mRNA levels of the type 2 IP(3) receptor were significantly decreased in all tested tissues, cerebellum, hypothalamus, and also in brain stem after the treatment with retinoic acid. These results show that gene expression of both type 1 and 2 IP(3) receptors is regulated by retinoic acid, although the effect of retinoic acid on mRNA levels of the type 1 IP(3) receptors is dependent on brain area.

It has been previously shown that modification of thyroid hormone levels have a profound impact on cardiac function, predominantly through a direct regulation of the sarcoplasmic reticulum protein levels. Nevertheless, little is known about the regulation of calcium transport systems in skeletal muscle due to the altered concentration of thyroid hormones. Thus, the goal of our study was to find out whether altered thyroid status could change the gene expression of the Na(+)/Ca(2+) exchanger (NCX), the inositol 1,4,5-trisphosphate (IP(3)) receptors and ryanodine receptors (RyRs) in slow and fast skeletal muscles of rats. A hyperthyroid state was maintained in rats by triiodothyronine (T(3)) administration, while methimazole was employed for inducing hypothyroidism. After a period of 2-10 months of T(3) treatment we observed a significant increase in mRNA levels of the NCX, RyRs and IP(3) receptors. This increase was more pronounced in the slow soleus than in the fast extensor digitorum longus (EDL) muscle. It is tempting to speculate that thyroid hormones also alter calcium concentration and thus influence the process of excitation-contraction coupling in the skeletal muscle.

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-s-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 2 h, 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/μg 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/μg 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 meausured 22 h after the termination of the stress stimulus. Repeated immobilization (7 days, 2 h 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, 2 h 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.

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.

Several cDNAs encoding an isoform of the α 1 subunit of the voltage-dependent calcium channel were isolated from rat brain cDNA libraries. The complete nucleotide sequence of 6975 by encodes a protein of 1634 amino acids, which corresponds to an M r of 186,968. The protein exhibits 71% and 76% homology to skeletal and cardiac α 1 subunits, respectively. When compared with skeletal and cardiac α 1 isoforms, the rat brain protein is intermediate in size at the amino terminus and shorter at the carboxyl terminus. Multiple subtypes of this α 1 isoform cDNA were characterized. These are indicative of alternative splicing of a primary transcript and encode three variants between motif I and motif II and two within the S3 region of motif IV. Thus, multiple isoforms of this rat brain α 1 subunit are possible.

Inositol 1,4,5-triphosphate receptors (IP3R) are intracellular calcium channels localised mainly on the endoplasmic reticulum. Up to now, three types of IP3R were characterised – type 1 (IP3R1), type 2 (IP3R2) and type 3 (IP3R3), differing predominantly in their localisation. These channels are releasing calcium upon binding IP3to the channel protein. Calcium release through IP3R can result in the activation of inner (mitochondrial) pathway of apoptosis induction. Thus, modulation of these channels in cancer cells is of a special importance. Unravelling regulation and function of IP3R, especially under the pathological situations, can result in the development of new therapeutic strategies based on the IP3R’s activation and/or blocking. Aim of the present work was to investigate an interaction between the calcium and sulfide signalling pathways, particularly effects of the slow H2S release donor morpholin-4-ium-4-methoxyphenyl-(morpholino)-phosphino-dithioate (GYY4137) on the expression of (IP3R) with the possible impact on the apoptosis induction in HeLa cells. Gene expression, Western blot analysis, apoptosis determination by Annexin V-Fluos and drop in mitochondrial membrane potential by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC1) and immunofluorescence were used to determine differences in control and GYY4137 treated HeLa cells. In HeLa cell line, GYY4137 (10 μmol L−1) up-regulated expression of the IP3R1 and IP3R2, but not IP3R3 on both, mRNA and protein levels. Concurrently, cytosolic calcium increased and reticular calcium was depleted in concentration-dependent manner, partially by the involvement of IP3R. Depletion of calcium from reticulum was accompanied by increase in endoplasmic reticulum (ER) stress markers, such as X-box, CHOP and ATF4, thus pointing to a development of ER stress due to GYY4137 treatment. Also, GYY4137 treatment of HeLa cells increased number of apoptotic cells. These results suggest an involvement of H2S in both IP3-induced calcium signalling and induction of apoptosis, possibly through the activation of ER stress.

Hydrogen sulfide (H2S) as a novel gasotransmitter regulates variety of processes, among them calcium transport systems. Sodium calcium exchanger (NCX) is one of key players in a regulation of calcium homeostasis. Thus, the aim of our work was to determine effect of sulfide signaling on the NCX type 1 (NCX1) expression and function in HeLa cells, to investigate relationship of beta-adrenergic receptors with the NCX1 in a presence and/or absence of H2S and to determine physiological importance of this potential communication. As a H2S donor we used morpholin-4-ium-4-methoxyphenyl (morpholino) phosphino-dithioate (GYY4137; 10 μ M). We observed increased levels of the NCX1 mRNA, protein and activity after 24 h of GYY4137 treatment. This increase was accompanied by elevated cAMP due to the GYY4137 treatment, which was completely abolished, when NCX1 was silenced. Increased cAMP levels would point to up-regulation of beta-adrenergic receptors. Indeed, GYY4137 increased expression of beta1 and beta3 (but not beta2) adrenergic receptors. These receptors co-precipitated, co-localized with the NCX1 and induced apoptosis in the presence of H2S. Our results suggest that sulfide signaling plays a role in the apoptosis induction through the up-regulation and mutual interaction of the NCX1, beta1 and beta3 adrenergic receptors, which might be of a potential importance in a cancer treatment. This work was supported by grants CEMAN and APVV51-0045-11.

The sodium calcium exchanger (NCX) is one of key players in the regulation of an intracellular calcium homeostasis. Although majority of papers dealing with the NCX1 comes from the cardiac tissue, importance of this calcium transporter was documented also in other types of cells and tissues, e.g. brain, endocrine system and also in immune system. Also, calcium influx through the reverse mode NCX1 is required for the activation and the targeting of proteinkinase C α to the plasma membrane, an essential step for vascular endothelial growth factor (VEGF) - induced ERK1/2 phosphorylation and downstream endothelial cells’ functions in angiogenesis. Nevertheless, little is known about the modulation as well as a role of the NCX1 in cancer cells. At least in cardiac tissue, signaling through the individual types of adrenergic receptors (AR) belongs to the most important regulations of the NCX1. Ncx1 is upregulated at both transcriptional and protein levels with β -AR stimulation in neonatal rat cardiomyocytes and in the adult rat heart. Therefore, our aim was to investigate the relationship of β -adrenergic receptors with the NCX1 in the presence and/or absence of H 2 S and to determine the physiological importance of the potential interaction in cancer cells. For our experiments we used a stable HeLa cell line, which is derived from epithelial cells of cervical cancer. As a H 2 S donor we used morpholin-4-ium-4-methoxyphenyl (morpholino) phosphinodithioate GYY4137. We have observed increased levels of the NCX1 mRNA, protein and activity after 24hours of GYY4137 treatment. Sulfide signaling also affect expression of the β 1 and β 3 adrenergic receptors and they co-immunoprecipitate and co-localize with the NCX1. GYY4137 increased number of Annexin V positive cells, which points to the development of apoptosis. GYY4137 induced apoptosis was abolished by NCX1 siRNA and also by β 1 and β 3, but not β 2 antagonists. These results suggest that by sulfide signaling NCX1, β 1 and β 3 AR co-localised and induced apoptosis, which might be of a potential importance in the cancer treatment.