6-(bromomethylene)tetrahydro-3-(1-naphthaleneyl)-2h-pyran-2-one and chelerythrine

Store-operated Ca2+ entry (SOCE) is an important mechanism for Ca2+ influx in smooth muscle cells; however the activation and regulation of this influx pathway are incompletely understood. In the present study we have examined the effect of several protein kinases in regulating SOCE in pulmonary artery smooth muscle cells (PASMCs) of the rat. Inhibition of protein kinase C with chelerythrine (3microM) potentiated SOCE by 47+/-2%, while the tyrosine kinase inhibitors genistein (100microM) and tyrphostin 23 (100microM) caused a significant reduction in SOCE of 55+/-9% and 43+/-7%, respectively. It has been proposed that Ca2+-insensitive phospholipase A(2) (iPLA(2)) is involved in the activation of SOCE in many different cell types. The iPLA(2) inhibitor, bromoenol lactone had no effect on SOCE, suggesting that this mechanism was not involved in the activation of the pathway. The calmodulin antagonists, calmidazolium (CMZ) (10microM) and W-7 (10microM) appeared to potentiate SOCE in PASMCs. Further investigation established that CMZ was actually activating a Ca2+ influx pathway that was independent of the filling state of the sarcoplasmic reticulum. The CMZ-activated Ca2+ influx was blocked by Gd3+ (10microM), but unaffected by 2-APB (75microM), indicating a pharmacological profile distinct from the classical SOCE pathway.

Topics: Animals; Benzophenanthridines; Boron Compounds; Calcium; Calmodulin; Cells, Cultured; Enzyme Inhibitors; Genistein; Imidazoles; Ion Transport; Male; Myocytes, Smooth Muscle; Naphthalenes; Phospholipases A2; Protein Kinase C; Protein-Tyrosine Kinases; Pulmonary Artery; Pyrones; Rats; Rats, Sprague-Dawley; Sulfonamides; Tyrphostins

The role of PKC in the regulation of store-operated Ca2+ entry (SOCE) is rather controversial. Here, we used Ca2+-imaging, biochemical, pharmacological, and molecular techniques to test if Ca2+-independent PLA2beta (iPLA2beta), one of the transducers of the signal from depleted stores to plasma membrane channels, may be a target for the complex regulation of SOCE by PKC and diacylglycerol (DAG) in rabbit aortic smooth muscle cells (SMCs). We found that the inhibition of PKC with chelerythrine resulted in significant inhibition of thapsigargin (TG)-induced SOCE in proliferating SMCs. Activation of PKC by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) caused a significant depletion of intracellular Ca2+ stores and triggered Ca2+ influx that was similar to TG-induced SOCE. OAG and TG both produced a PKC-dependent activation of iPLA2beta and Ca2+ entry that were absent in SMCs in which iPLA2beta was inhibited by a specific chiral enantiomer of bromoenol lactone (S-BEL). Moreover, we found that PKC regulates TG- and OAG-induced Ca2+ entry only in proliferating SMCs, which correlates with the expression of the specific PKC-epsilon isoform. Molecular downregulation of PKC-epsilon impaired TG- and OAG-induced Ca2+ influx in proliferating SMCs but had no effect in confluent SMCs. Our results demonstrate that DAG (or OAG) can affect SOCE via multiple mechanisms, which may involve the depletion of Ca2+ stores as well as direct PKC-epsilon-dependent activation of iPLA2beta, resulting in a complex regulation of SOCE in proliferating and confluent SMCs.

Topics: Animals; Benzophenanthridines; Calcium; Calcium Signaling; Cell Proliferation; Cells, Cultured; Diglycerides; Enzyme Activation; Enzyme Inhibitors; Group VI Phospholipases A2; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Naphthalenes; Protein Kinase C-epsilon; Pyrones; Rabbits; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Time Factors; Transfection