Yan-Ping Dai

ClC‐3 chloride channel is upregulated by hypertrophy and inflammation in rat and canine pulmonary artery

1 Cl− channels have been implicated in essential cellular functions including volume regulation, progression of cell cycle, cell proliferation and contraction, but the physiological functions of the ClC‐3 channel are controversial. We tested the hypothesis that the ClC‐3 gene (ClCn‐3) is upregulated in hypertensive pulmonary arteries of monocrotaline‐treated rats, and upregulated ClC‐3 channel aids viability of pulmonary artery smooth muscle cells (PASMCs). 2 Experimental pulmonary hypertension was induced in rats by a single subcutaneous administration of monocrotaline (60 mg kg−1). Injected animals developed characteristic features of pulmonary hypertension including medial hypertrophy of pulmonary arteries and right ventricular hypertrophy. 3 Reverse transcriptase–polymerase chain reaction (RT–PCR), immunohistochemistry and Western immunoblot analysis indicated that histopathological alterations were associated with upregulation of the ClC‐3 mRNA and protein expression in both smooth muscle cells of hypertensive pulmonary arteries and in cardiac myocytes. 4 RT–PCR analysis of mRNA, extracted from canine cultured PASMCs, indicated that incubation with the inflammatory mediators endothelin‐1 (ET‐1), platelet‐derived growth factor (PDGF), interleukin‐1beta (IL‐1β) and tumor necrosis factor alpha (TNFα), but not transforming growth factor beta (TGFβ), upregulated ClC‐3 mRNA. 5 Adenovirus‐mediated delivery and overexpression of ClC‐3 in canine PASMCs improved cell viability against increasing concentrations of hydrogen peroxide (H2O2, range 50–250 μM). 6 In conclusion, upregulation of ClC‐3 in rat hypertensive lung and heart is a novel observation. Our functional data suggest that upregulation of ClC‐3 is an adaptive response of inflamed pulmonary artery, which enhances the viability of PASMCs against reactive oxygen species.

Hypotonic Activation of Short ClC3 Isoform Is Modulated by Direct Interaction between Its Cytosolic C-terminal Tail and Subcortical Actin Filaments

Short ClC3 isoform (sClC3) functions as a volume-sensitive outwardly rectifying anion channel (VSOAC) in some cell types. In previous studies, we have shown that the hypotonic activation of sClC3 is linked to cell swelling-mediated remodeling of the actin cytoskeleton. In the present study, we have tested the hypothesis that the cytosolic tails of sClC3 bind to actin directly and that binding modulates the hypotonic activation of the channel. Co-sedimentation assays in vitro demonstrated a strong binding between the glutathione S-transferase-fused cytosolic C terminus of sClC3 (GST-sClC3-CT) to filamentous actin (F-actin) but not to globular monomeric actin (G-actin). The GST-fused N terminus (GST-sClC3-NT) exhibited low binding affinity to both G- and F-actin. Co-sedimentation experiments with progressively truncated GST-sClC3-CT indicated that the F-actin binding region is located between amino acids 690 and 760 of sClC3. Two synthetic peptides mapping basic clusters of the cytosolic sClC3-CT (CTP2, isoleucine 716 to leucine 734; and CTP3, proline 688 to proline 709) prevented binding of GST-sClC3-CT to F-actin in vitro. Dialysis into NIH/3T3 cells of these two peptides (but not of synthetic peptide CTP1 (isoleucine 737 to glutamine 748)) reduced the maximal current density by 60 and 38%, respectively. Based on these results, we have concluded that, by direct interaction with subcortical actin filaments, sClC3 contributes to the hypotonic stress-induced VSOACs in NIH/3T3 cells.

PDGF and IL-1β upregulate cofilin and LIMK2 in canine cultured pulmonary artery smooth muscle cells

Actin cytoskeleton reorganization is regulated by various actin-binding proteins. Cofilin is the principal filament-depolymerizing protein, whose activity is reduced upon phosphorylation by LIMK. Thus, LIMK and cofilin comprise a signal transduction module regulating actin turnover and myogenic tone in healthy vasculature. Novel functions of smooth muscle cells (SMCs) in the hypertensive pulmonary artery, such as increased motility and proliferation, are supported by the actin cytoskeleton. We therefore hypothesized that bioactive peptides that affect these SMC functions may also result in an upregulation of LIMK and cofilin expression. Semiquantitative RT-PCR and immunoblotting indicated that LIMK2 and cofilin mRNA and protein expression is upregulated in canine pulmonary artery SMCs (PASMCs) exposed to PDGF or IL-1β (10 ng/ml). Inhibition of ERK MAPKs (U-0126, 10 µM) or p38 MAPK (PD-169316, 10 µM), but not PI3Ks (LY-294002, 50 µM), reduced LIMK2 and cofilin gene expression stimulated by PDGF or IL-1β. Inhibition of ROCK (Y-27632, 10 µM) reduced only the IL-1β-stimulated LIMK2 and cofilin expression. These novel observations in PASMCs indicate that LIMK2 and cofilin expression can be induced by PDGF or IL-1β. This parallel upregulation of LIMK2 and cofilin may have potentially broad functional significance for the progress of pulmonary artery disease.

Storage and secretion of β-NAD, ATP and dopamine in NGF-differentiated rat pheochromocytoma PC12 cells

In nerve–smooth muscle preparations β‐nicotinamide adenine dinucleotide (β‐NAD) has emerged as a novel extracellular substance with putative neurotransmitter and neuromodulator functions. β‐NAD is released, along with noradrenaline and adenosine 5′‐triphosphate (ATP), upon firing of action potentials in blood vessels, urinary bladder and large intestine. At present it is unclear whether noradrenaline, ATP and β‐NAD are stored in and released from common populations of synaptic vesicles. The answer is unattainable in complex systems such as nerve–smooth muscle preparations. Adrenal chromaffin cells are thus used here as a single‐cell model to examine mechanisms of concomitant neurosecretion. Using high‐performance liquid chromatography techniques with electrochemical and fluorescence detection we simultaneously evaluated secretion of dopamine (DA), ATP, adenosine 5′‐diphosphate, adenosine 5′‐monophosphate, adenosine, β‐NAD and its immediate metabolites ADP‐ribose and cyclic ADP‐ribose in superfused nerve growth factor‐differentiated rat pheochromocytoma PC12 cells. β‐NAD, DA and ATP were released constitutively and upon stimulation with high‐K+ solution or nicotine. Botulinum neurotoxin A tended to increase the spontaneous secretion of all substances and abolished the high‐K+‐evoked release of β‐NAD and DA but not of ATP. Subcellular fractionation by continuous glycerol and sucrose gradients along with immunoblot analysis of the vesicular marker proteins synaptophysin and secretogranin II revealed that β‐NAD, ATP and DA are stored in both small synaptic‐like vesicles and large dense‐core‐like vesicles. However, the three substances appear to have different preferential sites of release upon membrane depolarization including sites associated with SNAP‐25 and sites not associated with SNAP‐25.

Hypotonic Activation of Volume-sensitive Outwardly Rectifying Anion Channels (VSOACs) Requires Coordinated Remodeling of Subcortical and Perinuclear Actin Filaments

Cell volume regulation requires activation of volume-sensitive outwardly rectifying anion channels (VSOACs). The actin cytoskeleton may participate in the activation of VSOACs but the roles of the two major actin pools remain undefined. We hypothesized that structural reorganization of both subcortical and perinuclear actin filaments (F-actin) contributes to the hypotonic activation of VSOACs. Hypotonic stress of pulmonary artery smooth muscle cells (PASMCs) was associated with reorganization of both peripheral and perinuclear F-actin, and with activation of VSOACs. Preincubation with cytochalasin D caused prominent dissociation of perinuclear, but not of subcortical F-actin. Cytochalasin D failed to induce isotonic activation and delayed the hypotonic activation of VSOACs. F-actin stabilization by phalloidin delayed both the hypotonic stress-induced dissociation of membrane-associated actin filaments and the activation kinetics of VSOACs. PKCε, which was proposed to phosphorylate and inhibit VSOACs, colocalized primarily with F-actin and the net kinase activity remained unchanged during hypotonic cell swelling. In conclusion, normal hypotonic activation of VSOACs requires disruption of peripheral F-actin but intact perinuclear F-actin; interference with this pattern of actin reorganization delays the activation kinetics of VSOACs. The cell swelling-induced peripheral actin dissociation may underlie the observed translocation of PKCε, which leads to a net decrease of PKCε inhibitory activity in submembranous sites. Thus, reorganization of actin and PKCε may establish conditions for mechano- and/or signal transduction-mediated activation of VSOACs.

Release, neuronal effects and removal of extracellular β-nicotinamide adenine dinucleotide (β-NAD+) in the rat brain

Recent evidence supports an emerging role of β‐nicotinamide adenine dinucleotide (β‐NAD+) as a novel neurotransmitter and neuromodulator in the peripheral nervous system –β‐NAD+ is released in nerve‐smooth muscle preparations and adrenal chromaffin cells in a manner characteristic of a neurotransmitter. It is currently unclear whether this holds true for the CNS. Using a small‐chamber superfusion assay and high‐sensitivity high‐pressure liquid chromatography techniques, we demonstrate that high‐K+ stimulation of rat forebrain synaptosomes evokes overflow of β‐NAD+, adenosine 5′‐triphosphate, and their metabolites adenosine 5′‐diphosphate (ADP), adenosine 5′‐monophosphate, adenosine, ADP‐ribose (ADPR) and cyclic ADPR. The high‐K+‐evoked overflow of β‐NAD+ is attenuated by cleavage of SNAP‐25 with botulinum neurotoxin A, by inhibition of N‐type voltage‐dependent Ca2+ channels with ω‐conotoxin GVIA, and by inhibition of the proton gradient of synaptic vesicles with bafilomycin A1, suggesting that β‐NAD+ is likely released via vesicle exocytosis. Western analysis demonstrates that CD38, a multifunctional protein that metabolizes β‐NAD+, is present on synaptosomal membranes and in the cytosol. Intact synaptosomes degrade β‐NAD+. 1,N 6‐etheno‐NAD, a fluorescent analog of β‐NAD+, is taken by synaptosomes and this uptake is attenuated by authentic β‐NAD+, but not by the connexin 43 inhibitor Gap 27. In cortical neurons local applications of β‐NAD+ cause rapid Ca2+ transients, likely due to influx of extracellular Ca2+. Therefore, rat brain synaptosomes can actively release, degrade and uptake β‐NAD+, and β‐NAD+ can stimulate postsynaptic neurons, all criteria needed for a substance to be considered a candidate neurotransmitter in the brain.