Sergiy G. Shlykov

Activation of Murine Lung Mast Cells by the Adenosine A3 Receptor

Adenosine has been implicated to play a role in asthma in part through its ability to influence mediator release from mast cells. Most physiological roles of adenosine are mediated through adenosine receptors; however, the mechanisms by which adenosine influences mediator release from lung mast cells are not understood. We established primary murine lung mast cell cultures and used real-time RT-PCR and immunofluorescence to demonstrate that the A2A, A2B, and A3 adenosine receptors are expressed on murine lung mast cells. Studies using selective adenosine receptor agonists and antagonists suggested that activation of A3 receptors could induce mast cell histamine release in association with increases in intracellular Ca2+ that were mediated through Gi and phosphoinositide 3-kinase signaling pathways. The function of A3 receptors in vivo was tested by exposing mice to the A3 receptor agonist, IB-MECA. Nebulized IB-MECA directly induced lung mast cell degranulation in wild-type mice while having no effect in A3 receptor knockout mice. Furthermore, studies using adenosine deaminase knockout mice suggested that elevated endogenous adenosine induced lung mast cell degranulation by engaging A3 receptors. These results demonstrate that the A3 adenosine receptor plays an important role in adenosine-mediated murine lung mast cell degranulation.

Antibodies From Preeclamptic Patients Stimulate Increased Intracellular Ca2+ Mobilization Through Angiotensin Receptor Activation

Background—Preeclampsia is a serious disorder of pregnancy characterized by hypertension, proteinuria, edema, and coagulation and vascular abnormalities. At the cellular level, abnormalities include increased calcium concentration in platelets, lymphocytes, and erythrocytes. Recent studies have shown that antibodies directed against angiotensin II type I (AT1) receptors are also highly associated with preeclampsia. Methods and Results—We tested the hypothesis that AT1 receptor–agonistic antibodies (AT1-AAs) could activate AT1 receptors, leading to an increased intracellular concentration of free calcium and to downstream activation of Ca2+ signaling pathways. Sera of 30 pregnant patients, 16 diagnosed with severe preeclampsia and 14 normotensive, were examined for the presence of IgG capable of stimulating intracellular Ca2+ mobilization. IgG from all preeclamptic patients activated AT1 receptors and increased intracellular free calcium. In contrast, none of the normotensive individuals had IgG capable of activating AT1 receptors. The specific mobilization of intracellular Ca2+ by AT1-AAs was blocked by losartan, an AT1 receptor antagonist, and by a 7-amino-acid peptide that corresponds to a portion of the second extracellular loop of the AT1 receptor. In addition, we have shown that AT1-AA–stimulated mobilization of intracellular Ca2+ results in the activation of the transcription factor, nuclear factor of activated T cells. Conclusions—These results suggest that maternal antibodies capable of activating AT1 receptors are likely to account for increased intracellular free Ca2+ concentrations and changes in gene expression associated with preeclampsia.

Multiple Trp Isoforms Implicated in Capacitative Calcium Entry Are Expressed in Human Pregnant Myometrium and Myometrial Cells

Abstract Capacitative Ca2+ entry plays a role in thapsigargin- and oxytocin-mediated increases in intracellular free Ca2+ in human myometrium. Members of the Trp protein family have been implicated in capacitative Ca2+ entry in a number of tissues. Pregnant human myometrium and the human myometrial cell line PHM1-41 expressed mRNA for hTrp1, hTrp3, hTrp4, hTrp6, and hTrp7. A number of known splice variants of hTrp1 and hTrp4 were expressed in these cells. In addition, novel splice variants for hTrp1 and hTrp3 were discovered. hTrp1γ1 and hTrp1γ2 contain insertions between previously described exons 9 and 10 that would alter reading frame and produce Trp proteins truncated in the membrane spanning region if expressed. The hTrp3 variant introduces sequence between exons 8 and 9 that would insert 16 amino acids in the C-terminal region of the protein upstream of the calmodulin and inositol 1,4,5-triphosphate receptor interaction domain. hTrp1, hTrp3, and hTrp4 proteins were detected in both pregnant human myometrial and PHM1-41 membranes; a weak band consistent with hTrp6 expression was detected in pregnant human myometrium. These data are consistent with the presence of proteins that could form putative capacitative Ca2+ channels in human myometrium. Control of the activity of these channels may be important for the control of uterine contractile activity.

Molecular signaling through G-protein-coupled receptors and the control of intracellular calcium in myometrium.

Cellular mechanisms regulating myometrial intracellular free calcium (Ca2+) 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 incerase in myosin light chain, an increase in myosin denosine monophosphatse (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 multuiple signaling pathways, including receptor-mediated activation of phospholipase Cβ (PLCβ), 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 those effects require association of protein kinase A (PKA) with a plasma membraneassociated 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, Trp Cs, 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.

Capacitative Cation Entry in Human Myometrial Cells and Augmentation by hTrpC3 Overexpression

Abstract Transient receptor potential (Trp) channels have been implicated in mediating store- and receptor-activated Ca2+ influx. Different properties of this influx in various cell types may stem from the assembly of these Trp proteins into homo- or heterotetramers or association with other regulatory proteins. We examined the properties of endogenous capacitative Ca2+ entry in PHM1 immortalized human myometrial cells that express endogenous hTrpCs 1, 3, 4, 6, and 7 mRNA and in primary human myocytes. In PHM1 cells, activation of the oxytocin receptor or depletion of intracellular Ca2+ stores with the endoplasmic reticulum calcium pump-inhibitor thapsigargin induced capacitative Ca2+ entry, which was inhibited both by SKF 96365 and gadolinium (Gd3+). Whereas unstimulated cells did not exhibit Sr2+ entry, oxytocin and thapsigargin enhanced Sr2+ entry that was also inhibited by SKF 96365 and Gd3+. In contrast, Ba2+, a poor substrate for Ca2+ pumps, accumulated in these cells in the absence of the capacitative entry stimulus and also after oxytocin and thapsigargin treatment. Both types of entry were markedly decreased by SKF 96365 and Gd3+. The membrane-permeant derivative of diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (OAG), elicited oscillatory increases in PHM1 intracellular Ca2+ that were dependent on extracellular Ca2+. These properties were also observed in primary human myocytes. Overexpression of hTrpC3 in PHM1 cells enhanced thapsigargin-, oxytocin-, and OAG-induced Ca2+ entry. These data are consistent with the expression of endogenous hTrpC activity in myometrium. Capacitative Ca2+ entry can potentially contribute to Ca2+ dynamics controlling uterine smooth muscle contractile activity.