PI3K and PKC contribute to membrane depolarization mediated by α(2)-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. EFS (0.1–2 Hz, 0.1 ms, 15V, 10 s)-induced changes in the membrane potential were recorded with a conventional intracellular microelectrode technique and evaluated in the absence and presence of inhibitors of neuronal activity, α-adrenoceptors, membrane ion channels, PI3K, inositol 1,4,5-triphosphate (InsP3) receptors, and PKC. Activation of PI3Kγ and PKCζ in response to exogenous NE and clonidine in the absence and presence of receptor and kinase inhibitors were also determined. RESULTS: Contractile responses to NE and clonidine (0.05 – 10 μM) were significantly diminished in the presence of yohimbine (0.1 μM). Exogenous NE (0.1 μM) and clonidine (1 μM) elicited SMD. The resting membrane potential of canine mesenteric vein smooth muscle cells was -68.8 ± 0.8 mV. EFS elicited a biphasic depolarization comprised of excitatory junction potentials and SMD that are purinergic and adrenergic in nature, respectively. The magnitude of the SMD in response to EFS at 0.5 Hz was 9.4 ± 0.7 mV. This response was reduced by 65–98% by the fast Na(+ )channel inhibitor tetrodotoxin (1 μM), by the inhibitor of N-type Ca(2+ )channels ω-conotoxin GVIA (5 nM), the non-selective α-adrenoceptor blocker phentolamine (1 μM), the selective α(2)-adrenoceptor blocker yohimbine (0.1 μM), the ion channel inhibitors niflumic acid (NFA, 100 μM), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 30 μM), 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 200 μM), and Gd(3+ )(30 μM), and the PI3K inhibitors wortmannin (100 nM) and LY-294002 (10 μM). The SMD remained unchanged in the presence of the L-type Ca(2+ )channel blocker nicardipine (1 μM) and the InsP(3 )receptor blockers 2-aminoethoxydiphenylborate (2APB, 50 μM) and xestospongin C (3 μM). The inhibitor of PKC chelerythrine (1 μM), but not calphostin C (10 μM), diminished the SMD. Exogenous NE and clonidine (1 μM each) activated both PI3Kγ and PKCζ, and the activation of these kinases was abolished by preincubation of tissue with the α(2)-adrenoceptor blocker yohimbine. CONCLUSION: Neuronally-released NE stimulates smooth muscle α(2)-adrenoceptors and activates PI3K and atypical PKC in the canine mesenteric vein. Events downstream of PKC lead to SMD and vasoconstriction. This represents a novel pathway for NE-induced membrane depolarization in a vascular smooth muscle preparation

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 22.5% in PA and differentiated PASMCs, but only 14.8% in undifferentiated PASMCs. With comparable contraction responses in PA, endothelin-1 (100 nM) and norepinephrine (1 μM) induced a 2-fold increase of P-CF/CF, while angiotensin II (1 μM) induced none. All agonists activated Rho-kinase and LIMK2, and activation was eliminated by inhibition of Rho-kinase. Microcystin LF (20 nM) potentiated the angiotensin II, but not the 5-hydroxytryptamine (1 μM)-mediated increase of P-CF/CF. In conclusion, all tested agonists activate the Rho-kinase-LIMK pathway and increase P-CF/CF. Angiotensin II activates PP2A and counteracts the LIMK-mediated CF phosphorylation. CF phosphorylation stabilizes peripheral actin structures and may contribute to the maximal contraction of PA

NAD+-metabolizing ecto-enzymes shape tumor–host interactions: The chronic lymphocytic leukemia model

AbstractNicotinamide adenine dinucleotide (NAD+) is an essential co-enzyme that can be released in the extracellular milieu. Here, it may elicit signals through binding purinergic receptors. Alternatively, NAD+ may be dismantled to adenosine, up-taken by cells and transformed to reconstitute the intracellular nucleotide pool. An articulated ecto-enzyme network is responsible for the nucleotide–nucleoside conversion. CD38 is the main mammalian enzyme that hydrolyzes NAD+, generating Ca2+-active metabolites. Evidence suggests that this extracellular network may be altered or used by tumor cells to (i) nestle in protected areas, and (ii) evade the immune response. We have exploited chronic lymphocytic leukemia as a model to test the role of the ecto-enzyme network, starting by analyzing the individual elements that make up the whole picture

P2Y receptors as regulators of lung endothelial barrier integrity

Endothelial cells (ECs), forming a semi-permeable barrier between the interior space of blood vessels and underlying tissues, control such diverse processes as vascular tone, homeostasis, adhesion of platelets, and leukocytes to the vascular wall and permeability of vascular wall for cells and fluids. Mechanisms which govern the highly clinically relevant process of increased EC permeability are under intense investigation. It is well known that loss of this barrier (permeability increase) results in tissue inflammation, the hall mark of inflammatory diseases such as acute lung injury and its severe form, acute respiratory distress syndrome. Little is known about processes which determine the endothelial barrier enhancement or protection against permeability increase. It is now well accepted that extracellular purines and pyrimidines are promising and physiologically relevant barrier-protective agents and their effects are mediated by interaction with cell surface P2Y receptors which belong to the superfamily of G-protein-coupled receptors. The therapeutic potential of P2Y receptors is rapidly expanding field in pharmacology and some selective agonists became recently available. Here, we present an overview of recently identified P2Y receptor agonists that enhance the pulmonary endothelial barrier and inhibit and/or reverse endothelial barrier disruption

NUDT2 Disruption Elevates Diadenosine Tetraphosphate (Ap4A) and Down-Regulates Immune Response and Cancer Promotion Genes.

Regulation of gene expression is one of several roles proposed for the stress-induced nucleotide diadenosine tetraphosphate (Ap4A). We have examined this directly by a comparative RNA-Seq analysis of KBM-7 chronic myelogenous leukemia cells and KBM-7 cells in which the NUDT2 Ap4A hydrolase gene had been disrupted (NuKO cells), causing a 175-fold increase in intracellular Ap4A. 6,288 differentially expressed genes were identified with P < 0.05. Of these, 980 were up-regulated and 705 down-regulated in NuKO cells with a fold-change ≥ 2. Ingenuity® Pathway Analysis (IPA®) was used to assign these genes to known canonical pathways and functional networks. Pathways associated with interferon responses, pattern recognition receptors and inflammation scored highly in the down-regulated set of genes while functions associated with MHC class II antigens were prominent among the up-regulated genes, which otherwise showed little organization into major functional gene sets. Tryptophan catabolism was also strongly down-regulated as were numerous genes known to be involved in tumor promotion in other systems, with roles in the epithelial-mesenchymal transition, proliferation, invasion and metastasis. Conversely, some pro-apoptotic genes were up-regulated. Major upstream factors predicted by IPA® for gene down-regulation included NFκB, STAT1/2, IRF3/4 and SP1 but no major factors controlling gene up-regulation were identified. Potential mechanisms for gene regulation mediated by Ap4A and/or NUDT2 disruption include binding of Ap4A to the HINT1 co-repressor, autocrine activation of purinoceptors by Ap4A, chromatin remodeling, effects of NUDT2 loss on transcript stability, and inhibition of ATP-dependent regulatory factors such as protein kinases by Ap4A. Existing evidence favors the last of these as the most probable mechanism. Regardless, our results suggest that the NUDT2 protein could be a novel cancer chemotherapeutic target, with its inhibition potentially exerting strong anti-tumor effects via multiple pathways involving metastasis, invasion, immunosuppression and apoptosis