The lack of a calcium channel agonist (e.g., BayK8644) for CaV2 channels has impeded their investigation. Roscovitine, a potent inhibitor of cyclin-dependent kinases 1, 2, and 5, has recently been reported to slow the deactivation of P/Q-type calcium channels (CaV2.1). We show that roscovitine also slows deactivation (EC(50) approximately 53 microM) of N-type calcium channels (CaV2.2) and investigate gating alterations induced by roscovitine. The onset of slowed deactivation was rapid ( approximately 2 s), which contrasts with a slower effect of roscovitine to inhibit N-current (EC(50) approximately 300 microM). Slow deactivation was specific to roscovitine, since it could not be induced by a closely related cyclin-dependent kinase inhibitor, olomoucine (300 microM). Intracellularly applied roscovitine failed to slow deactivation, which implies an extracellular binding site. The roscovitine-induced slow deactivation was accompanied by a slight left shift in the activation-voltage relationship, slower activation at negative potentials, and increased inactivation. Additional data showed that roscovitine preferentially binds to the open channel to slow deactivation. A model where roscovitine reduced a backward rate constant between two open states was able to reproduce the effect of roscovitine on both activation and deactivation.

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Thus, our study offers the first evidence that nNav contribute to arrhythmogenic DCR, thereby providing a conceptual basis for mechanism-based antiarrhythmic therapy.

To determine the role of phosphoinositide 3-OH kinase (PI3K) pathways in the acute vascular permeability increase associated with ventilator-induced lung injury, we ventilated isolated perfused lungs and intact C57BL/6 mice with low and high peak inflation pressures (PIP). In isolated lungs, filtration coefficients ( Kf) increased significantly after ventilation at 30 cmH2O (high PIP) for successive periods of 15, 30 (4.1-fold), and 50 (5.4-fold) min. Pretreatment with 50 μM of the PI3K inhibitor, LY-294002, or 20 μM PP2, a Src kinase inhibitor, significantly attenuated the increase in Kf, whereas 10 μM Akt inhibitor IV significantly augmented the increased Kf. There were no significant differences in Kf or lung wet-to-dry weight (W/D) ratios between groups ventilated with 9 cmH2O PIP (low PIP), with or without inhibitor treatment. Total lung β-catenin was unchanged in any low PIP isolated lung group, but Akt inhibition during high PIP ventilation significantly decreased total β-catenin by 86%. Ventilation of intact mice with 55 cmH2O PIP for up to 60 min also increased lung vascular permeability, indicated by increases in lung lavage albumin concentration and lung W/D ratios. In these lungs, tyrosine phosphorylation of β-catenin and serine/threonine phosphorylation of Akt, glycogen synthase kinase 3β (GSK3β), and ERK1/2 increased significantly with peak effects at 60 min. Thus mechanical stress activation of PI3K and Src may increase lung vascular permeability through tyrosine phosphorylation, but simultaneous activation of the PI3K-Akt-GSK3β pathway tends to limit this permeability response, possibly by preserving cellular β-catenin.

Lung vascular permeability is acutely increased by high-pressure and high-volume ventilation. To determine the roles of mechanically activated cytosolic PLA2 (cPLA2)and Clara cell secretory protein (CCSP), a modulator of cPLA2 activity, we compared lung injury with and without a PLA2 inhibitor in wild-type mice and CCSP-null mice (CCSP-/-) ventilated with high and low peak inflation pressures (PIP) for 2- or 4-h periods. After ventilation with high PIP, we observed significant increases in the bronchoalveolar lavage albumin concentrations, lung wet-to-dry weight ratios, and lung myeloperoxidase in both genotypes compared with unventilated controls and low-PIP ventilated mice. All injury variables except myeloperoxidase were significantly greater in the CCSP-/- mice relative to wild-type mice. Inhibition of cPLA2 in wild-type and CCSP-/- mice ventilated at high PIP for 4 h significantly reduced bronchoalveolar lavage albumin and total protein and lung wet-to-dry weight ratios compared with vehicle-treated mice of the same genotype. Membrane phospho-cPLA2 and cPLA2 activities were significantly elevated in lung homogenates of high-PIP ventilated mice of both genotypes but were significantly higher in the CCSP-/- mice relative to the wild-type mice. Inhibition of cPLA2 significantly attenuated both the phospho-cPLA2 increase and increased cPLA2 activity due to high-PIP ventilation. We propose that mechanical activation of the cPLA2 pathway contributes to acute high PIP-induced lung injury and that CCSP may reduce this injury through inhibition of the cPLA2 pathway and reduction of proinflammatory products produced by this pathway.