Bryophyllum pinnatum Compounds Inhibit Oxytocin-Induced Signaling Pathways in Human Myometrial Cells

Bryophyllum pinnatum has been used in the treatment of premature labor, first in anthroposophic hospitals and, recently, in conventional settings as an add-on medication. In vitro work with hTERT human myometrial cells showed that B. pinnatum leaf press juice inhibits the increase of intracellular free calcium concentration induced by oxytocin, a hormone known to play a role in labor. Our aim was to identify fractions/compounds in B. pinnatum press juice that contribute to this inhibitory effect, and to investigate their effect on oxytocin-driven activation of the MAPK cascade. Several fractions/compounds from B. pinnatum press juice led to a concentration-dependent decrease of oxytocin-induced increase of intracellular free calcium concentration, but none of them was as strong as B. pinnatum press juice. However, the combination of a bufadienolide and a flavonoid-enriched fraction was as effective as B. pinnatum press juice, and their combination had a synergistic effect. B. pinnatum press juice inhibited oxytocin-driven activation of MAPKs SAPK/JNK and ERK1/2, an effect also exerted by the bufadienolide-enriched fraction. The effect of B. pinnatum press juice on oxytocin-induced signaling pathways was comparable to that of the oxytocin-receptor antagonist and tocolytic agent atosiban. Our findings further substantiate the use of B. pinnatum press juice preparations in the treatment of preterm labor.

SAH-Induced Electrophysiological Changes of Ventricular Myocytes and Role of N-acetylcysteine Protection

Background Electrocardiogram (ECG) changes in patients with subarachnoid hemorrhage (SAH) are frequent. ST- and/or T-wave changes in ECG seem to predominate. Study Aims To investigate the ion channel mechanisms of SAH-induced ventricular excitation-contraction coupling changes and the possible protective effect of N-acetylcysteine (NAC). Methods Three groups of rabbits were used for the experiments. In two groups, SAH was induced by replacing the cerebrospinal fluid (CSF) with fresh autologous blood. In the control group, CSF was replaced with isotonic saline. In one SAH group, NAC was administered daily beginning at SAH induction. On day 5, ventricular action potentials, ionic currents, contractions, and intracellular free ion concentrations were recorded from the myocytes. Results In the SAH group, no change was found in the sodium currents, but the transient outward potassium currents were depressed, rapid repolarizing currents were increased, and t-type calcium currents were increased. Contractions and the intracellular free calcium concentration were depressed. NAC treatment, in contrast, not only restores these electrical remodeling changes but also the contractile abnormalities in the cardiac myocytes. Conclusion The changes in the action potential duration can be attributed to the measured ionic current changes. However, the exact mechanism, other than the oxidative stress, by which the NAC treatment protects the cardiac muscle needs additional investigations.

Endothelial mitochondria regulate the intracellular Ca2+ response to fluid shear stress

Shear stress is known to stimulate an intracellular free calcium concentration ([Ca2+]i) response in vascular endothelial cells (ECs). [Ca2+]i is a key second messenger for signaling that leads to vasodilation and EC survival. Although it is accepted that the shear-induced [Ca2+]i response is, in part, due to Ca2+ release from the endoplasmic reticulum (ER), the role of mitochondria (second largest Ca2+ store) is unknown. We hypothesized that the mitochondria play a role in regulating [Ca2+]i in sheared ECs. Cultured ECs, loaded with a Ca2+-sensitive fluorophore, were exposed to physiological levels of shear stress. Shear stress elicited [Ca2+]i transients in a percentage of cells with a fraction of them displaying oscillations. Peak magnitudes, percentage of oscillating ECs, and oscillation frequencies depended on the shear level. [Ca2+]i transients/oscillations were present when experiments were conducted in Ca2+-free solution (plus lanthanum) but absent when ECs were treated with a phospholipase C inhibitor, suggesting that the ER inositol 1,4,5-trisphosphate receptor is responsible for the [Ca2+]i response. Either a mitochondrial uncoupler or an electron transport chain inhibitor, but not a mitochondrial ATP synthase inhibitor, prevented the occurrence of transients and especially inhibited the oscillations. Knockdown of the mitochondrial Ca2+ uniporter also inhibited the shear-induced [Ca2+]i transients/oscillations compared with controls. Hence, EC mitochondria, through Ca2+ uptake/release, regulate the temporal profile of shear-induced ER Ca2+ release. [Ca2+]i oscillation frequencies detected were within the range for activation of mechanoresponsive kinases and transcription factors, suggesting that dysfunctional EC mitochondria may contribute to cardiovascular disease by deregulating the shear-induced [Ca2+]i response.

189 CHARACTERIZATION OF THE FIRST SPERM-INDUCED CALCIUM TRANSIENT IN PIG OOCYTES

Fertilization in mammals is associated with repetitive elevations in the oocytes’ intracellular free calcium concentration. The elevations are triggered by the fertilizing sperm and are responsible for stimulating embryo development. In mouse oocytes, the sperm-induced calcium signal starts with a calcium rise that is larger and longer in duration than any succeeding transients. It also has unique characteristics: it begins with a rapid increase for 2–3 s followed by a shoulder, which is an inflection point that represents a brief decline in the rise of calcium levels. Once calcium level reaches its maximum, it decreases but remains elevated for several minutes while it is superimposed by several smaller calcium spikes. In bovine oocytes the situation is somewhat different. In this species, the first sperm-induced calcium transient is larger than the additional spikes but it lacks the sustained elevation phase and is not superimposed by small calcium rises. In the present study our purpose was to characterise the first sperm-induced calcium transient in pig oocytes. Oocytes were obtained from ovaries of prepubertal gilts collected at an abattoir and matured in vitro for 44 h. Mature oocytes were loaded with the calcium indicator dye fura-2; subsequently, they were either IVF or used for intracytoplasmic sperm injection (ICSI). Changes in their intracellular free calcium concentration were then immediately monitored using InCyt Im2, a dual-wavelength fluorescence imaging system. Characteristics of the first transients (including amplitude and duration) were compared to those of the additional ones using Student’s t-test. We found that in oocytes that underwent IVF (n = 11), the oscillations started 83.4 ± 23.2 min after adding the sperm to the oocytes. In the ICSI group (n = 10 oocytes) the calcium oscillations started sooner, 27.1 ± 17.7 min after injection. The average peak amplitude and the mean interval between the calcium transients varied among individual oocytes, but no significant differences were found between the IVF and ICSI groups (which on average were fluorescence ratio of 2.6 ± 1.1 and 23.5 ± 11.4 min, respectively; P > 0.1). The oscillation patterns showed slight differences between individual oocytes in terms of spike frequency, which has been described before and may be due to variations in the amount of sperm-derived activating factor present in the ooplasm. Most importantly, in all oocytes measured, the initial calcium spike showed no differences when compared to subsequent calcium transients: its amplitude and duration was similar to the additional transients. This points at potential species-specific differences in the regulation of calcium signalling in oocytes and provides essential information for the better understanding of the fertilization process. This work was supported by Agriculture and Food Research Initiative Competitive Grant 2011–67015–30006 from the USDA National Institute of Food and Agriculture.

Petchienes A–E, Meroterpenoids from Ganoderma petchii

Petchienes A-E (1–5), five new meroterpenoids, were isolated from the fruiting bodies of Ganoderma petchii. Their structures, including absolute configurations, were elucidated by means of spectroscopic and computational methods. Compound 4 was isolated as a racemic mixture, which was finally purified by chiral HPLC to yield individual (-) and (+)-antipodes. Biological evaluation showed that compounds 2 and (-)-4 could increase intracellular free calcium concentration at 10 μM in HEK-293 cells.

Intracellular Calcium Plays a Critical Role in the Microcystin-LR-Elicited Neurotoxicity Through PLC/IP3 Pathway

Neurotoxicity of microcystin-leucine-arginine (MCLR) has been widely reported. However, the mechanism is not fully understood. Using primary hippocampal neurons, we tested the hypothesis that MCLR-triggered activation in intracellular free calcium concentration ([Ca2+]i) induces the death of neurons. Microcystin-leucine-arginine inhibited cell viability at a range of 0.1 to 30 μmol/L and caused a dose-dependent increase in [Ca2+]i. This increase in [Ca2+]i was observed in Ca2+-free media and blocked by an endoplasmic reticulum Ca2+ pump inhibitor, suggesting intracellular Ca2+ release. Moreover, pretreatment of hippocampal neurons with intracellular Ca2+ chelator (O,O′-bis (2-aminophenyl) ethyleneglycol-N,N,N′,N′-tetraacetic acid, tetraacetoxy-methyl ester) and inositol 1,4,5-trisphosphate receptor antagonist (2-aminoethoxydiphenyl borate) could block both the Ca2+ mobilization and the neuronal death following MCLR exposure. In contrast, the ryanodine receptor inhibitor (dantrolene) did not ameliorate the effect of MCLR. In conclusion, MCLR disrupts [Ca2+]i homeostasis in neurons by releasing Ca2+ from intracellular stores, and this increase in [Ca2+]i may be a key determinant in the mechanism underlying MCLR-induced neurotoxicity.