α2-Adrenoceptor signaling in cardiomyocytes of spontaneously hypertensive rats starts to impair already at early age.

α2-Adrenoceptors (α2-AR) found in the cardiomyocyte's sarcolemma represent a very important negative feedback for control of myocardial contractility by endogenous catecholamines. Earlier, we showed that the endogenous neurotransmitter agmatine in micromolar concentrations via α2-AR activates the nitric oxide (NO) synthesis, enhancing the Ca2+ pumping into sarcoplasmic reticulum (SR). In the millimolar doses it inhibits Ca2+ sequestration by SR Ca2+ ATPase (SERCA), acting through the first type of imidazoline receptors. Here, we study the functional activity of agmatine, as well as a specific α2-agonist, guanabenz, in respect to spontaneous Ca2+-transients in SHR cardiomyocytes of the early age (2-2.5 months), and adulthood animals (8-9 months). α2-mediated cardioprotective effect was almost twofold decreased in SHR cardiac cells compared to normotensive rats of the corresponding age, despite the fact that both α2A- and α2B-AR protein levels were significantly increased in SHR cardiomyocytes. NO-mediated facilitation of SERCA activity is substantially reduced in SHR cardiomyocytes vs. normotensive rats. These data suggest that the SHR phenotype starting from early age shows signs of the impaired sarcolemmal α2-AR signaling, which can aggravate the development of this cardiovascular pathology.

Synergism of myocardial ß-adrenoceptor blockade and I1-imidazoline receptor-driven signaling: Kinase-phosphatase switching.

Recently identified imidazoline receptors of the first type (I1Rs) on the cardiomyocyte's sarcolemma open a new field in calcium signaling research. In particular, it is interesting to investigate their functional interaction with other well-known systems, such as ß-adrenergic receptors. Here we investigated the effects of I1Rs activation on L-type voltage-gated Ca2+-currents under catecholaminergic stress induced by the application of ß-agonist, isoproterenol. Pharmacological agonist of I1Rs (I1-agonist), rilmenidine, and the putative endogenous I1-ligand, agmatine, have been shown to effectively reduce Ca2+-currents potentiated by isoproterenol. Inhibitory analysis shows that the ability to suppress voltage-gated Ca2+-currents by rilmenidine and agmatine is fully preserved in the presence of the protein kinase A blocker (PKA), which indicates a PKA-independent mechanism of their action. The blockade of NO synthase isoforms with 7NI does not affect the intrinsic effects of agmatine and rilmenidine, which suggests NO-independent signaling pathways triggered by I1Rs. A nonspecific serine/threonine protein phosphatase (STPP) inhibitor, calyculin A, abrogates effects of rilmenidine or agmatine on the isoproterenol-induced Ca2+-currents. Direct measurements of phosphatase activity in the myocardial tissues showed that activation of the I1Rs leads to stimulation of STPP, which could be responsible for the I1-agonist influences. Obtained data clarify peripheral effects that occur during activation of the I1Rs under endogenous catecholaminergic stress, and can be used in clinical practice for more precise control of heart contractility in some cardiovascular pathologies.

[Changes of mitochondrial DNA/nuclear DNA ratio in the blood serum following X-ray irradiation of mice at various doses].

Using quantitative real-time PCR, the copy number of mitochondrial and nuclear DNA fragments in mouse blood serum was estimated at different time points following X-ray irradiation at various doses (from 0.5 to 10 Gy). The changes in the correlation between mtDNA and nuclear DNA (mtDNA/nucDNA) in blood serum reflect the degree of radiation injury depending on the dose of irradiation. Exposure to radiation at 10 Gy and massive cell death caused by this lethal dose result in a sharp decrease by an order of magnitude of the mtDNA/nucDNA ratio in the mouse serum; the value of this parameter did not recover within the next 3 days. The opposite effect was revealed when mice were exposed to irradiation at the dose of I Gy, which is not followed by massive cell death, but leads to a higher level of the mtDNA damage as compared with the nuclear DNA protected by histones. Defective mtDNA molecules enter the bloodstream, which results in an increase of the mtDNA/nucDNA ratio in serum. Under irradiation of mice at the intermediate dose of 3 Gy the two processes described above are exhibited at once. During the first hours after irradiation an apoptotic death of radiosensitive cells and release of a large number of nuclear DNA fragments in the serum are initiated, which reduces the mtDNA/nucDNA ratio. However, at later times after irradiation, starting from 5 days, an increase of the mtDNA/nucDNA ratio is observed in the serum, presumably as a result of reparation and elimination of defective mtDNA. Thus, the mtDNA/nucDNA ratio in the serum of irradiated mice reflects the degree of the radiation damage to cells and may be considered as a biological marker of radiation injury in the future.

[Mitochondrial genetic apparatus functioning in mice spleen cells under radiation-induced apoptosis].

An important role of mitochondria in the process of programmed cell death is widely accepted now. There is a set of nuclear-encoded mitochondrial proteins involved in this process. Apart from this, a mitochondrion contains its own genetic apparatus comprising mtDNA and replication, transcription, and translation systems. However, a mechanism of mitochondria genetic information realization under apoptosis-inducing conditions has been understood poorly. Here, using the real-time PCR technique the number of mitochondrial genes and their transcripts in mouse spleen cells after whole-body X-ray irradiation at the dosage of 10 Gy has been evaluated. During 5 h after the irradiation a nuclear DNA was subjected to fragmentation, whereas mtDNA remained intact. Moreover, in the course of time after irradiation the number of mtDNA copies increased threefold. A process of mtDNA transcription was more susceptible to the irradiation: in 1 h after exposure the number of ND2, ND4 and CYTB gene transcripts were sharply decreased. In 24 and 72 h after the irradiation the amount of ND2 and ND4 transcripts was restored to the control values, while the CYTB one remained low; the number of ATP6 transcripts was compared with the control within the whole period of observation. The difference in levels of mRNAs for the genes transcribed under the control of the same promoter for mice to be grown both under normal conditions and after x-ray irradiation allows us to propose the existing a posttranscriptional mechanism which regulates expression of mitochondrial genes and provides different recovery rates for different mitochondrial transcripts at the development of apoptosis.

[Sharp changes in the copy number of mtDNA and its transcription in the blood cells of X-ray irradiated mice are observed, and mtDNA fragments appear in the blood serum].

Changes in the number of mitochondrial DNA (mtDNA) copies and alterations in its transcription were followed in the blood cells of mice after their exposure to X-rays; also, extracellular mtDNA fragments were registered in the blood serum of these mice. By a real time polymerase chain reaction (RT-PCR), a sharp increase in mtDNA copy number one hour after the exposure was detected in the blood cells of 1 Gy X-ray irradiated mice. This increase in mtDNA copies is considered as a result of a compensatory reaction developed in mice in response to the radiation damage in a part of mtDNA molecules. Exposure to X-ray radiation at a dose of 10 Gy resulted in an arrest of mtDNA replication in mouse blood cells within three hours after irradiation. At the same time, RT-PCR assays showed that at the same radiation dose, an increase in the number of mtRNA of the mitochondrial nd6 gene relative to mRNA of the nuclear gene gapdh occurs. This indicates that, although mtDNA is more damaged in comparison with nuclear DNA (nDNA), the process of transcription continues in the blood cell mitochondria of mice after their exposure to 10 Gy, whereas the transcription of damaged nDNA is arrested. After the irradiation of mice with 10 Gy, mtDNA fragments of 1841 bp were registered in the blood serum. Thus, in the blood cells of mice exposed to X-rays, the copy number of mtDNA and its transcription are sharply changed, and large mtDNA fragments are observed in the blood serum.

[Release of mtDNA from mitochondria and activation of its replication in tissues of irradiated mice].

A nessessary condition for normal functioning of mitochondria is the maintenance of certain numbers of intact mtDNA molecules. In the present study, we investigasted changes in the number of mtDNA copies in brain and spleen cells of mice subjected to irradiation. For the first time, we observed the irradiation-induced output of mtDNA fragments into brain and spleen cell cytosol. In the cytosol of these cells, examined in mice 5 h after 5 Gy irradiation, 1841 h.p. mtDNA fragments were detected able to persist for at 3 weeks. In addition, larger fragments of mtDNA (10,090 b.p.) were detected in the cytosol of brain cells of irradiated mice. The occurrence of mtDNA fragments in the cytosol of brain cells is accompanied with an increase in the number of mtDNA copies in the mitochondrial matrix. The induction of mtDNA replication in brain cells of irradiated animals may be considered as a compensatory reaction in response to mtDNA damage. A sharp decrease in the amount of mtDNA copies in the mitochondrial matrix of spleen cells on the first day after irradiation may be considered as apoptosis development. However, the compensatory reaction in brain cells was also noticed but in later terms.