Pulmonary vascular medial hypertrophy due to proliferation of pulmonary artery smooth muscle cells (PASMC) greatly contributes to the increased pulmonary vascular resistance in pulmonary hypertension patients. A rise in cytosolic free Ca2+ concentration ([Ca2+]cyt) is an important stimulus for cell growth in PASMC. Resting [Ca2+]cyt, intracellularly stored [Ca2+], capacitative Ca2+ entry (CCE), and store-operated Ca2+ currents (I(SOC)) are greater in proliferating human PASMC than in growth-arrested cells. Expression of TRP1, a transient receptor potential gene proposed to encode the channels responsible for CCE and I(SOC), was also upregulated in proliferating PASMC. Our aim was to determine if inhibition of endogenous TRP1 gene expression affects I(SOC) and CCE and regulates cell proliferation in human PASMC. Cells were treated with an antisense oligonucleotide (AS, for 24 h) specifically designed to cleave TRP1 mRNA and then returned to normal growth medium for 40 h before the experiments. Then, mRNA and protein expression of TRP1 was downregulated, and amplitudes of I(SOC) and CCE elicited by passive depletion of Ca2+ from the sarcoplasmic reticulum using cyclopiazonic acid were significantly reduced in the AS-treated PASMC compared with control. Furthermore, the rate of cell growth was decreased by 50% in AS-treated PASMC. These results indicate that TRP1 may encode a store-operated Ca2+ channel that plays a critical role in PASMC proliferation by regulating CCE and intracellular [Ca2+](cyt).

No abstract

SUMMARYThe ketogenic diet (KD) is an effective treatment for epilepsy, but its mechanisms of action are poorly understood. We investigated the hypothesis that the KD inhibits mammalian target of rapamycin (mTOR) pathway signaling. The expression of pS6 and pAkt, markers of mTOR pathway activation, was reduced in hippocampus and liver of rats fed KD. In the kainate model of epilepsy, KD blocked the hippocampal pS6 elevation that occurs after status epilepticus. Because mTOR signaling has been implicated in epileptogenesis, these results suggest that the KD may have anticonvulsant or antiepileptogenic actions via mTOR pathway inhibition.

Agonist-induced increases in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in pulmonary artery (PA) smooth muscle cells (SMCs) consist of a transient Ca(2+) release from intracellular stores followed by a sustained Ca(2+) influx. Depletion of intracellular Ca(2+) stores triggers capacitative Ca(2+) entry (CCE), which contributes to the sustained increase in [Ca(2+)](cyt) and the refilling of Ca(2+) into the stores. In isolated PAs superfused with Ca(2+)-free solution, phenylephrine induced a transient contraction, apparently by a rise in [Ca(2+)](cyt) due to Ca(2+) release from the intracellular stores. The transient contraction lasted for 3-4 min until the Ca(2+) store was depleted. Restoration of extracellular Ca(2+) in the presence of phentolamine produced a contraction potentially due to a rise in [Ca(2+)](cyt) via CCE. The store-operated Ca(2+) channel blocker Ni(2+) reduced the store depletion-activated Ca(2+) currents, decreased CCE, and inhibited the CCE-mediated contraction. In single PASMCs, we identified, using RT-PCR, five transient receptor potential gene transcripts. These results suggest that CCE, potentially through transient receptor potential-encoded Ca(2+) channels, plays an important role in agonist-mediated PA contraction.