Cellular mechanisms regulating myometrial intracellular free calcium (Ca2+(i)) are addressed in this review, with emphasis on G-protein-coupled receptor pathways. An increase in myometrial Ca2+(i) results in phosphorylation of myosin light chain, an increase in myosin adenosine monophosphatase (ATPase) activity and contraction. Dephosphorylation of myosin light chain and a decline in Ca2+(i) are associated with relaxation. Increases in Ca2+(i) are controlled by multiple signaling pathways, including receptor-mediated activation of phospholipase Cbeta (PLCbeta), leading to release of Ca2+ from intracellular stores. Ca2+ also enters myometrial cells through plasma membrane Ca2+ channels. Conversely, adenosine triphosphate (ATP)-dependent Ca2+ pumps lower Ca2+(i) concentrations and potassium channels promote hyperpolarization that can decrease Ca2+ entry. Receptor-coupled pathways that promote uterine relaxation primarily involve activation of cyclic adenosine monophosphate (cAMP)- or cyclic guanosine monophosphate (cGMP)-stimulated protein kinases that phosphorylate proteins regulating Ca2+ homeostasis. cAMP has inhibitory effects on myometrial contractile activity, agonist-stimulated phosphatidylinositide turnover and increases in Ca2+(i). Some of these effects require association of protein kinase A (PKA) with a plasma membrane-associated A-kinase-anchoring-protein (AKAP). Near term in the rat, there is a decline in the plasma membrane localization of PKA associated with this anchoring protein. This correlates with changes in the regulation of signaling pathways controlling Ca2+(i). L-type voltage-operated Ca2+ entry is an important regulator of myometrial contraction. In addition, putative signal-regulated or capacitative Ca2+ channel proteins, TrpCs, are expressed in myometrium, and signal-regulated Ca2+ entry is observed in human myometrial cells. This Ca2+ entry mechanism may play a significant role in the control of myometrial Ca2+(i) dynamics and myometrial contraction. The regulation of myometrial Ca2+(i) is complex. Understanding the mechanisms involved may lead to design of tocolytics that target multiple pathways and achieve improved suppression of premature labor.

External Ca2+ entry into myometrial smooth-muscle cells is important to uterine contraction and hence to labor progression and parturition. Proteins of the transient receptor potential (Trp) channel family are putative capacitative Ca2+ entry channels that respond to contractant-generated signals and intracellular Ca2+ store depletion. Quantitative reverse transcription-polymerase chain reaction was used to examine the relative expression of TrpC mRNAs in rat myometrium and determine their expression pattern during pregnancy and labor. rTrpC1, rTrpC2, rTrpC4, rTrpC5, rTrpC6, and rTrpC7 mRNAs, but not rTrpC3 mRNA, were expressed in nonpregnant rat myometrium. With the exception of rTrpC7, the resulting products were sequenced and found to be identical with published sequences; new rTrpC7 sequence exhibited >88% homology to mouse and human TrpC7 coding regions. Relative to beta-actin mRNA, rTrpC4 mRNA was expressed in the greatest abundance. rTrpC1, 5, and 6 mRNAs were expressed at lower levels, whereas rTrpC2 and 7 mRNAs were barely detectable. This relative expression pattern was also observed throughout the course of gestation. There were no major differences in expression of rTrpC1, 2, 4, or 7 mRNAs between Day 13 and Day 21 of gestation or labor. Rat TrpC5 and TrpC6 mRNA expression decreased in pregnancy but was not altered between Day 13 and Day 21 or in labor. Western blot analysis generally confirmed these observations with respect to protein expression. These data suggest that rTrpC4 may play a major role in regulated Ca2+ entry in myometrial cells and throughout pregnancy but do not rule out contributions from other Trp proteins.

We have demonstrated that hTrpC1 and hTrpC4 are the most abundant TrpC mRNAs in human myometrium, with TrpC6 being the next most abundant. There was no increase in TrpC mRNA or protein in fundal myometrium with the onset of labor. Nonetheless, these isoforms may play significant roles in signal regulated calcium entry in human myometrium.

No abstract

To examine ryanodine-sensitive Ca channels in mitochondria of rat hepatocytes and their role in energy state of the cells via investigation of the ryanodine effect on mitochondrial membrane potential. Oxygen consumption was measured by polarography using the Clark electrode. The substrates of oxidation such as pyruvate (5mM), α-ketoglutarate (5mM), or succinate (5mM) were used. Oxidative phosphorylation was stimulated by the addition of adenosine diphosphate (200nM). Mitochondrial membrane potential was measured using a voltage-sensitive fluorescent probe tetramethylrhodamine-methyl-ester (0.1μM) and was analyzed by a flow cytometer. To evaluate the intact mitochondria, we used carbonil cyanide m-chlorophenyl hydrazone (CCCP, 10μM). Changes in the ionized calcium concentration in rat liver mitochondria were measured using a fluorescent probe Fluo-4 AM. Effect of ryanodine on oxygen consumption of rat liver mitochondria depends on the oxidation substrate and the incubation time. Oxidation of pyruvate in the presence of ryanodine (0.05μM) decreased the membrane potential of rat liver mitochondria by 38.4%. At higher concentrations, ryanodine (0.1μM or 1μM) led to decrease of membrane potential by 51.7% and 42.8%, respectively. In contrast, oxidation of α-ketoglutarate in the presence of ryanodine (0.05μM) increased mitochondrial membrane potential by 16.8%. However, at higher concentrations, ryanodine (0.1μM or 1μM) triggered a decreasing of membrane potential by 42.5% and 31.0%, respectively. Therefore, ryanodine at various concentrations (0.05μM, 0.1μM, or 1μM) causes differential effects on Ca concentration in the mitochondria matrix under oxidation of pyruvate or α-ketoglutarate. The data suggest the presence of ryanodine receptors in mitochondrial membrane of rat hepatocytes. Their inhibition with higher concentrations of ryanodine leads to decreasing of intra-mitochondrial Ca concentration and affecting the energy state of mictochondria in hepatocytes.

Calixarenes--supramolecular compounds interacting with bioactive molecules and ions that causes the changes in biochemical and biophysical processes. The aim of this work was to study the effects of calix[4]arenes C-136, C-137 and C-138 on the level of polarization of rat myometrium mitochondria membrane. Structure of synthesized calix[4]arene molecules was confirmed by the methods of 1H NMR and infra-red spectroscopy. Calix[4]arenes C-136 and C-137 possess two chalcone amide moieties at the lower rim, while the calix[4]arene C-138--only one. In case of calix[4]arenes C-136 and C-137 take place, accordingly, absence or presence of phenolic hydroxyl groups at the lower rim on the calix[4]arene skeleton. It was shown that calix[4]arenes C-136, C-137 and C-138 form micelles in a water medium and in the dimethylformamide (DMF). The irradiation of micelles with argon laser on flow cytometer results in appearance of autofluorescence. In the water medium calix[4]arene micelles interact with positively charged potential-sensitive fluorescent probe TMRM, that can testify to the presence of negative charge in these structures. However calix[4]arene micelles in DMF solution do not interact with TMRM. Mitochondrial membrane potential was measured using fluorescent dyes MTG and TMRM with confocal microscopy and fluorescent dye TMRM with flow cytometry. Experiments were conducted on myometrium cells in culture and on suspension of digitonin-permeabilized uterus myocytes. It was shown that a fluorescent signal was stable during time of experiment. Calix[4]arenes C-136 and C-137 (10 μM) hyperpolarize mitochondria membranes. A maximal effect was 173%. At the same time calix[4]arene C-138 did not influence on mitochondria membrane potential. Connection comes into question between structural organization of investigated calix[4]arene molecules and their influence on polarization of mitochondria membrane.

No abstract

Mitochondria are a key player in a wide range of the most important functions of the cell. calixarenes are supramolecular compounds that have been widely used in bioorganic chemistry and biochemistry. the aim of this work was to study the effects of calix[4]arenes with two (С-1012, С-1021), three (С-1023, С-1024) and four (С-1011) chalcone amide groups on the myometrial mitochondria membranes polarization, Ca 2+ concentration in the matrix of these organelles ([Ca 2+ ] m) and on the average hydrodynamic diameter of mitochondria. It was shown that permeabilized myometrium cells incubation with calix[4]arenes containing two or more chalcone amide groups, was accompanied by an increased level of myometrial mitochondria membranes polarization. All studied calix[4]arenes increased [Ca 2+ ] m values in the absence and in the presence of exogenous ca 2+. The values of [Ca 2+ ] m in the absence of exogenous ca 2+ were higher at mitochondria incubation in Mg 2+-containing, than in Mg 2+ ,Atp-containing medium. Incubation of isolated mitochondria with the studied calix[4]arenes resulted in changes of mitochondria volume: at incubation with С-1012, С-1021, C-1023 the average hydrodynamic diameter was decreased, while with С-1011 it was increased. Thus, we have shown that a short-term (5 min) incubation of mitochondria in the presence of 10 µM calix[4]arenes, which contain from two to four chalcone amide groups, increased the level of mitochondria membranes polarization, ionized Ca concentration in the matrix and had different effects on the mitochondrial volume. K e y w o r d s: myometrium, mitochondria, [Ca 2+ ] m , Mg 2+ , ATP, calix[4]arene chalcone amides. M itochondria play a significant role in a wide range of the most important functions of the cell, such as Са 2+ signaling, apoptosis, adaptation to stress, steroidogenesis, and aging [1, 2]. Disruption of the mitochondria functioning is a key element in age-dependent diseases, neurodegenerative disorders and various forms of cancer [2]. Channels and transporters of ions and metabolites through the mitochondrial membrane were directly involved in the regulation of mitochondrial functions and control of the cell metabolism. The capacity of the mitochondria to accumulate the Ca 2+ is known from 60 years of XX century [3], but the molecular mechanisms and significance of the Ca 2+ accumulation in the mitochondria have been clarified much later [4]. An increase in ioni zed Ca