Heterologous expression of the transient receptor potential-1 gene product (Trp1) encodes for a Ca2+ entry pathway, though it is unclear whether endogenous Trp1 contributes to a selective store-operated Ca2+ entry current. We examined the role of Trp1 in regulating both store-operated Ca2+ entry and a store-operated Ca2+ entry current, I(SOC), in A549 and endothelial cells. Twenty different 'chimeric' 2'-O-(2-methoxy)ethylphosphothioate antisense oligonucleotides were transfected separately using cationic lipids and screened for their ability to inhibit Trp1 mRNA. Two hypersensitive regions were identified, one at the 5' end of the coding region and the second in the 3' untranslated region beginning six nucleotides downstream of the stop codon. Antisense oligonucleotides stably decreased Trp1 at concentrations ranging from 10 to 300 nM, for up to 72 h. Thapsigargin increased global cytosolic Ca2+ and activated a I(SOC), which was small (-35 pA @ -80 mV), reversed near +40 mV, inhibited by 50 microM La3+, and exhibited anomalous mole fraction dependence. Inhibition of Trp1 reduced the global cytosolic Ca(2+) response to thapsigargin by 25% and similarly reduced I(SOC) by 50%. These data collectively support a role for endogenously expressed Trp1 in regulating a Ca2+-selective current activated upon Ca2+ store depletion.
Activation of Ca2+ entry is known to produce endothelial cell shape change, leading to increased permeability, leukocyte migration, and initiation of angiogenesis in conduit-vessel endothelial cells. The mode of Ca2+ entry regulating cell shape is unknown. We hypothesized that activation of store-operated Ca2+ channels (SOCs) is sufficient to promote cell shape change necessary for these processes. SOC activation in rat pulmonary arterial endothelial cells increased free cytosolic Ca2+ that was dependent on a membrane current having a net inward component of 5.45 ± 0.90 pA/pF at −80 mV. Changes in endothelial cell shape accompanied SOC activation and were dependent on Ca2+ entry-induced reconfiguration of peripheral (cortical) filamentous actin (F-actin). Because the identity of pulmonary endothelial SOCs is unknown, but mammalian homologues of the Drosophila melanogaster transient receptor potential ( trp) gene have been proposed to form Ca2+ entry channels in nonexcitable cells, we performed RT-PCR using Trp oligonucleotide primers in both rat and human pulmonary arterial endothelial cells. Both cell types were found to express Trp1, but neither expressed Trp3 nor Trp6. Our study indicates that 1) Ca2+ entry in pulmonary endothelial cells through SOCs produces cell shape change that is dependent on site-specific rearrangement of the microfilamentous cytoskeleton and 2) Trp1 may be a component of pulmonary endothelial SOCs.
Mechanism(s) underlying activation of store-operated Ca2+ entry currents, I SOC, remain incompletely understood. F-actin configuration is an important determinant of channel function, although the nature of interaction between the cytoskeleton and I SOC channels is unknown. We examined whether the spectrin membrane skeleton couples Ca2+ store depletion to Ca2+ entry. Thapsigargin activated an endothelial cell I SOC (−45 pA at −80 mV) that reversed at +40 mV, was inwardly rectifying when Ca2+ was the charge carrier, and was inhibited by La3+ (50 μM). Disruption of the spectrin–protein 4.1 interaction at residues A207-V445 of βSpIIΣ1 decreased the thapsigargin-induced global cytosolic Ca2+ response by 50% and selectively abolished the endothelial cell I SOC, without altering activation of a nonselective current through cyclic nucleotide–gated channels. In contrast, disruption of the spectrin–actin interaction at residues A47-K186 of βSpIIΣ1 did not decrease the thapsigargin-induced global cytosolic Ca2+ response or inhibit I SOC. Results indicate that the spectrin–protein 4.1 interaction selectively controls I SOC, indicating that physical coupling between calcium release and calcium entry is reliant upon the spectrin membrane skeleton.
The purpose of this review is to examine whether our current knowledge of the higher order control of gene expression and nuclear organization can help us understand the mechanisms of genomic imprinting. Imprinting involves the inheritance of a silenced allele of a gene through either a paternal or maternal germline. We have approached the problem of imprinting using a model based on the dynamic attachment of chromatin loops to immobilized RNA polymerases and control elements. We have combined the information from different experimental approaches, examining primarily the IGF2-H19 locus, in an attempt to simplify the complexity of the imprinting data that has accumulated. It is hoped that a unified model may generate predictions amenable to experimental testing and contribute to the interpretation of future experiments.
Calcium agonists induce membrane depolarization in endothelial cells through an unknown mechanism. Present studies tested the hypothesis that pulmonary artery endothelial cells express a cyclic nucleotide-gated (CNG) cation channel activated by store-operated calcium entry to produce membrane depolarization. In the whole-cell configuration, voltage-clamped cells revealed a large non-inactivating, outwardly rectifying cationic current in the absence of extra-or intracellular Ca 2؉ that was reduced upon replenishment of Ca 2؉ . The inward current was non-selective for K ؉ , Na ؉ , Cs ؉ , and Rb ؉ and was not inhibited by high tetraethylammonium concentrations. cAMP and cGMP stimulated the current and changed the cation permeability to favor Na ؉ . Moreover, 8-bromo-cAMP stimulated the current in voltageclamped cells in the perforated patch mode. The cationic current was inhibited by the CNG channel blocker LY83,583, and reverse transcriptase-polymerase chain reaction cloning identified expression of a CNG channel resembling that seen in olfactory neurons. Activation of store-operated calcium entry using thapsigargin increased a current through the CNG channel. Stimulation of the current paralleled pulmonary artery endothelial cell membrane depolarization, and both the current and membrane depolarization were abolished using LY83,583. Taken together, these data demonstrate activation of store-operated calcium entry stimulates a CNG channel producing membrane depolarization. Such membrane depolarization may contribute to slow feedback inhibition of store-operated calcium entry.
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