GSK3 regulates the function of mitochondrial proteins such as adenine nucleotide translocator (ANT) and cyclophilin D [9,10,11]. mitochondrial swelling was accelerated in the presence of the anti-CNPase antibody. H89 enhanced the effect of anti-CNPase antibody and accelerated the swelling of mitochondria, while CmZ abolished the effect of anti-CNPase antibody under mPTP opening. The levels of phospho-Akt and phospho-GSK3 increased, while the MEL content did not change. It can be assumed that CNPase may be involved in the regulation of these kinases, which in turn plays an important role in mPTP functioning. Keywords:permeability transition pore; protein phosphorylation; serine/threonine kinases; calmodulin; 2,3-cyclic nucleotide 3-phosphodiesterase; Rifaximin (Xifaxan) melatonin == 1. Introduction == The protein phosphorylation/dephosphorylation system regulates many cell functions and is involved in signal transduction. It can significantly influence the structural and morphological features of protein [1]. Many kinases and phosphatases responsible for phosphorylation and dephosphorylation, Rifaximin (Xifaxan) respectively, accomplish their functions in the cell primarily outside mitochondria but it is usually known that many protein kinases, phosphatases, and related proteins are also contained inside mitochondria [1]. One of the main protein kinases found in mitochondria Rifaximin (Xifaxan) is usually serine/threonine-activated kinase Akt, also known as protein kinase B [2]. Several studies showed that mitochondrial Akt catalyzes a phosphorylation and inactivates the pro-apoptotic protein BAD, and the recruitment of Raf-1 to the mitochondria promotes cell survival [3,4]. Recent investigations discovered new targets for mitochondrial Akt such as Complex V of the mitochondrial electron transport chain and hexokinase-II [5,6]. Akt also has a Rabbit polyclonal to GNMT direct effect in mitochondria, which is usually mediated by phosphorylation of hexokinase-II, resulting in the protection of mitochondria from oxidants or the opening of the Ca2+-induced mitochondrial permeability transition pore (mPTP) [6]. The mPTP opening results in the inhibition of respiration, mitochondrial swelling, the inner membrane depolarization, and the release of pro-apoptotic factors [7]. Another serine/threonine kinase, GSK3, is present in mitochondria of the rat cerebellum [8]. GSK3 regulates the function of mitochondrial proteins such as adenine nucleotide translocator (ANT) and cyclophilin D [9,10,11]. It is known that this inhibition of GSK leads to a lag in the opening of mPTP in response to oxygen radicals [12]. It has been suggested that this mPTP opening is usually a causative event in cell death during myocardial ischemia/reperfusion impairment [13,14]. GSK3 is usually localized in the cytosol and the nucleus but its action focuses on mitochondrial proteins; therefore, the knowledge of its function is usually of great interest. It was reported that the content of total and phosphorylated GSK3 in mitochondria of isolated rat hearts subjected to ischemia/reperfusion increases, and the proteinprotein conversation between GSK3 and VDAC or ANT enhances [15]. Phosphorylated GSK3 inhibits mPTP opening by a variety of mechanisms [16,17]. Ca2+, as a second messenger, might regulate the activity of different protein kinases and protein phosphatases through binding with calmodulin (CaM) [18]. CaM is usually a Ca2+-binding protein, which interacts with a great number of target proteins and regulates their functions. CaM is also found in great amounts in the nervous system where it interacts most intensively with the myelin basic protein, which has been confirmed by structural studies [19,20,21,22]. Recently a neuroprotective protein has been identified as 2,3-cyclic nucleotide-3-phosphodiesterase (CNPase) in rat brain mitochondria (RBM), and it has been shown that CNPase protects mitochondria from mPTP opening [23]. It is known that CNPase (CNP, EC 3.1.4.37) catalyzes the hydrolysis of 2,3-cyclic nucleotides to form the corresponding nucleoside 2-monophosphates [24]. The functions of CNPase in mitochondria are presently poorly comprehended. CNPase substrates (2,3-cAMP and 2,3-cNADP) were found to be able to accelerate mPTP opening in RBM as well as in rat liver mitochondria (RLM) [23], which indicates potential conversation of CNPase with the modulators of mPTP in mitochondria. CNPase interacts with RNA, CaM, and the cytoskeleton proteins [25]. It also inhibits translation to modulate mitochondrial membrane permeability and has putative ATP/GTPase activity. The addition of the anti-CNPase antibody to a mitochondrial suspension was found to protect mitochondria from mPTP opening [26,27]. CNPase is usually phosphorylated by protein kinase A (PKA) and protein kinase C [28]. The hormone of the pineal gland melatonin (N-acetyl-5-methoxytryptamine, MEL) is usually a highly conserved molecule found in a variety of organisms, from unicellular organisms to vertebrates [29,30]. Two isoforms of the melatonin receptor, MT1 and MT2, were found in the brain and peripheral tissues of mammals [31,32,33]. Both isoforms are expressed in brain mitochondria. It is believed that this interactions of MEL with its receptors may be involved in mitochondrial cell death, although melatonin (MEL) may also have other, non-receptor-mediated targets. MT1 was reported to be contained predominantly.