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.

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

In nerve-smooth muscle preparations beta-nicotinamide adenine dinucleotide (beta-NAD) has emerged as a novel extracellular substance with putative neurotransmitter and neuromodulator functions. beta-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 beta-NAD are stored in and released from common populations of synaptic vesicles.

Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 K-bZIP modulates latency-associated nuclear protein-mediated suppression of lytic origin-dependent DNA synthesis.

The original cotransfection replication assay identified eight human herpesvirus 8 (HHV8)-encoded proteins required for origin-dependent lytic DNA replication. Previously, we demonstrated that under conditions where K-Rta is overexpressed, a K-bZIP knockout bacmid displayed an aberrant subcellular localization pattern for the latency-associated nuclear protein (LANA). Additionally, these same studies demonstrated that K-bZIP interacts with LANA in the absence of K-Rta and that K-bZIP does not directly participate in, but may facilitate, the initiation of lytic DNA synthesis.

Cardiac-specific overexpression of the human short CLC-3 chloride channel isoform in mice.

1. ClC-3 has been proposed as a molecular candidate responsible for volume-sensitive outwardly rectifying anion channels (VSOAC) in cardiac and smooth muscle cells. To further test this hypothesis, we produced a novel line of transgenic mice with cardiac-specific overexpression of the human short ClC-3 isoform (hsClC-3).

N-type and P/Q-type calcium channels regulate differentially the release of noradrenaline, ATP and beta-NAD in blood vessels.

Using HPLC techniques we evaluated the electrical field stimulation-evoked overflow of noradrenaline (NA), adenosine 5'-triphosphate (ATP), and beta-nicotinamide adenine dinucleotide (beta-NAD) in the presence of low nanomolar concentrations of omega-conotoxin GVIA or omega-agatoxin IVA in the canine mesenteric arteries and veins. omega-conotoxin GVIA abolished the evoked overflow of NA and beta-NAD in artery and vein, whereas the evoked overflow of ATP remained unchanged in the presence of omega-conotoxin GVIA. omega-agatoxin IVA significantly reduced the evoked overflow of ATP and beta-NAD.

Distinct effects of contraction agonists on the phosphorylation state of cofilin in pulmonary artery smooth muscle.

We hypothesized that agonist-induced contraction correlates with the phospho-cofilin/cofilin (P-CF/CF) ratio in pulmonary artery (PA) rings and cultured smooth muscle cells (PASMCs). PA rings were used for isometric contractions and along with PASMCs for assay of P-CF/CF by isoelectric focusing and immunoblotting. The P-CF/CF measured 22.

Transcriptional repression of K-Rta by Kaposi's sarcoma-associated herpesvirus K-bZIP is not required for oriLyt-dependent DNA replication.

Kaposi's sarcoma-associated herpesvirus origin-dependent DNA replication requires the core replication proteins plus K-Rta and K-bZIP. To determine which K-bZIP protein domains contribute to oriLyt-dependent DNA replication and facilitate suppression of K-Rta-mediated transcriptional activation, we generated a series of deletion constructs and site-directed mutations within the K-bZIP ORF. Mutation of key leucine residues within the putative leucine zipper (LZ) motif eliminated the ability of the protein to homodimerize and complement oriLyt-dependent DNA replication.

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.

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.

Novel localization of CD38 in perivascular sympathetic nerve terminals.

Using high performance liquid chromatography fraction analysis we have recently established that numerous smooth muscle preparations, including the canine mesenteric artery and vein, release beta-nicotinamide adenine dinucleotide upon short-pulse electrical field stimulation in tetrodotoxin- and omega-conotoxin GVIA-sensitive manners [ Release of beta-nicotinamide adenine dinucleotide upon stimulation of postganglionic nerve terminals in blood vessels and urinary bladder. J Biol Chem 279:48893-48903.].

Upregulation of profilin, cofilin-2 and LIMK2 in cultured pulmonary artery smooth muscle cells and in pulmonary arteries of monocrotaline-treated rats.

Pulmonary hypertension is associated with remodeling of the smooth muscle layer of pulmonary arteries, manifested by reduced smooth muscle cell (SMC) contractility and enhanced motility and growth. These responses are underlied by increased dynamics of the peripheral actin network. Thus, we hypothesized that pulmonary hypertension is associated with upregulation of two proteins that regulate the dynamics of peripheral actin filaments, i.

beta-NAD is a novel nucleotide released on stimulation of nerve terminals in human urinary bladder detrusor muscle.

Endogenous nucleotides with extracellular functions may be involved in the complex neural control of human urinary bladder (HUB). Using HPLC techniques with fluorescence detection, we observed that in addition to ATP and its metabolites ADP, AMP and adenosine, electrical field stimulation (EFS; 4-16 Hz, 0.1 ms, 15 V, 60 s) of HUB detrusor smooth muscle coreleases novel nucleotide factors, which produce etheno-1N(6)-ADP-ribose (eADPR) on etheno-derivatization at high temperature.

PI3K and PKC contribute to membrane depolarization mediated by alpha2-adrenoceptors in the canine isolated mesenteric vein.

Background: Norepinephrine (NE), a classic neurotransmitter in the sympathetic nervous system, induces vasoconstriction of canine isolated mesenteric vein that is accompanied by a sustained membrane depolarization. The mechanisms underlying the NE-elicited membrane depolarization remain undefined. In the present study we hypothesized that phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) are involved in the electrical field stimulation (EFS)-induced slow membrane depolarization (SMD) in canine isolated mesenteric vein.

Human cytomegalovirus UL84 is a phosphoprotein that exhibits UTPase activity and is a putative member of the DExD/H box family of proteins.

Human cytomegalovirus (HCMV) UL84 is required for lytic DNA replication and is proposed to be the key factor in initiation of viral DNA synthesis. We now show that UL84 has a high degree of homology to the DExD/H (where x can be any amino acid) box family of helicases, displays UTPase activity, and is phosphorylated at serine residues. Affinity column-purified UL84-FLAG fusion protein was used in an in vitro nucleoside triphosphatase (NTPase) assay to show that UL84 has NTPase activity, preferring UTP.

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

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). Experimental pulmonary hypertension was induced in rats by a single subcutaneous administration of monocrotaline (60 mg kg(-1)).

PDGF and IL-1beta 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.

Hypotonic activation of volume-sensitive outwardly rectifying chloride channels in cultured PASMCs is modulated by SGK.

The serum- and glucocorticoid-inducible kinase (SGK) is a serine/threonine protein kinase (PK) transcriptionally regulated by corticoids, serum, and cell volume. SGK regulates cell volume of various cells by effects on Na(+) and K(+) transport through membrane channels. We hypothesized a role for SGK in the activation of volume-sensitive osmolyte and anion channels (VSOACs) in cultured canine pulmonary artery smooth muscle cells (PASMCs).

Functional effects of novel anti-ClC-3 antibodies on native volume-sensitive osmolyte and anion channels in cardiac and smooth muscle cells.

Whether ClC-3 encodes volume-sensitive organic osmolyte and anion channels (VSOACs) remains controversial. We have shown previously that native VSOACs in some cardiac and vascular myocytes were blocked by a commercial anti-ClC-3 carboxy terminal antibody (Alm C592-661 antibody), although recent studies have raised questions related to the specificity of Alm C592-661 antibody. Therefore, we have developed three new anti-ClC-3 antibodies and investigated their functional effects on native VSOACs in freshly isolated canine pulmonary artery smooth muscle cells (PASMCs) and guinea pig cardiac myocytes.

Canine mesenteric artery and vein convey no difference in the content of major contractile proteins.

Background: Mesenteric arteries and veins are composed of tonic smooth muscles and serve distinct functions in the peripheral circulation. However, the basis for the functional disparity of the resistive and capacitative parts of the mesenteric circulation is poorly understood. We studied potential differences in the expression levels of six contractile proteins in secondary and tertiary branches of the inferior mesenteric artery and vein along with differences in the vessel wall morphology.

Regulation of volume-sensitive outwardly rectifying anion channels in pulmonary arterial smooth muscle cells by PKC.

We tested the possible role of endogenous protein kinase C (PKC) in the regulation of native volume-sensitive organic osmolyte and anion channels (VSOACs) in acutely dispersed canine pulmonary artery smooth muscle cells (PASMC). Hypotonic cell swelling activated native volume-regulated Cl(-) currents (I(Cl.vol)) which could be reversed by exposure to phorbol 12,13-dibutyrate (0.

PI 3-kinases and Src kinases regulate spreading and migration of cultured VSMCs.

Pulmonary artery smooth muscle cell (PASMC) adhesion, spreading, and migration depend on matrix-stimulated reorganization of focal adhesions. Platelet-derived growth factor (PDGF) activates intracellular signal transduction cascades that also regulate adhesion, spreading, and migration, but the signaling molecules involved in these events are poorly defined. We hypothesized that phosphatidylinositol (PI) 3-kinases and Src tyrosine kinases translate matrix and PDGF-initiated signals into cell motility.

Modulatory role of ERK MAPK-caldesmon pathway in PDGF-stimulated migration of cultured pulmonary artery SMCs.

Extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs) phosphorylate caldesmon in vivo, but the function of caldesmon phosphorylation in smooth muscle physiology is controversial. We hypothesized that ERK MAPKs and caldesmon modulate chemotactic migration of cultured canine pulmonary artery smooth muscle cells (PASMCs). Platelet-derived growth factor (PDGF; 10 ng/ml) and endothelin-1 (ET-1; 100 nM) transiently activated ERK MAPKs: PDGF produced higher maximal and more potent activation of ERK MAPKs over 5 h.

Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody.

Intracellular dialysis of NIH/3T3 cells with a commercially available anti-ClC-3 polyclonal antibody (Ab) for approximately 30 min completely inhibited expressed guinea-pig ClC-3 currents (IgpClC-3), while intracellular dialysis with antigen-preabsorbed anti-ClC-3 Ab failed to affect IgpClC-3. Anti-ClC-3 Ab was used as a selective probe to examine the relationship between endogenous ClC-3 expression and native volume-sensitive outwardly rectifying anion channels (VSOACs) in guinea-pig cardiac cells, canine pulmonary arterial smooth muscle cells (PASMCs) and Xenopus laevis oocytes. Intracellular dialysis or injection of anti-ClC-3 Ab abolished native VSOAC function in cardiac cells and PASMCs and significantly reduced VSOACs in oocytes.

Phosphorylation of caldesmon by ERK MAP kinases in smooth muscle.

Phosphorylation of h-caldesmon has been proposed to regulate airway smooth muscle contraction. Both extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases phosphorylate h-caldesmon in vitro. To determine whether both enzymes phosphorylate caldesmon in vivo, phosphorylation-site-selective antibodies were used to assay phosphorylation of MAP kinase consensus sites.

Phosphatidylinositol 3-kinases regulate ERK and p38 MAP kinases in canine colonic smooth muscle.

In canine colon, M2/M3 muscarinic receptors are coupled to extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases. We tested the hypothesis that this coupling is mediated by enzymes of the phosphatidylinositol (PI) 3-kinase family. RT-PCR and Western blotting demonstrated expression of two isoforms, PI 3-kinase-alpha and PI 3-kinase-gamma.