
O-1 LOCALIZATION OF PANNEXIN 1 TO PLASMA MEMBRANE CAVEOLIN-1 SUPPORTS ATP RELEASE AND BLOOD PRESSURE REGULATION Leon J. DeLalio1,2, Alexander S. Keller1,2, Andrew Boyce4, Ms. Rachael Weaver2, Leigh Anne Swayne4, Richard Minshall5, Brant E. Isakson2,3 1Department of Pharmacology, University of Virginia, United States, 2Robert M. Berne Cardiovascular Research Center, University of Virginia, United States, 3Department of Molecular Physiology and Biophysics, University of Virginia, United States, 4Division of Medical Sciences, Centre for Biomedical Research, University of Victoria, Canada, 5Department of Pharmacology, University of Illinois at Chicago, United States Sympathetic nerve innervation to vascular smooth muscle cell (VSMCs) is a major regulator of arterial vasoconstriction and peripheral resistance. Importantly, the localization of α1-adrenergic receptors (α1-ARs) to the plasma membrane scaffold protein, Caveolin-1, is crucial for efficiently activating downstream signaling molecules important for vasoconstriction. Our lab has previously demonstrated that Pannexin 1 channels, which are highly expressed in VSMCs of resistance arteries, functionally couple with α1-AR, but not other vasoconstriction pathways, through extracellular release of ATP. We hypothesize that a unique Pannexin1 signaling domain exists on the plasma membrane of VSMCs that utilizes Caveolin-1 to localize Pannexin1 to areas of sympathetic innervation. We find here that Pannexin1 and αa1-ARs co-localize using proximity ligation assays at areas of sympathetic nerve innervation with VSMC plasma membrane. Importantly, these areas are enriched for caveolae. Using live cell imaging in primary VSMC culture and proximity ligation assays in intact resistance arteries, we also observed a novel interaction between Pannexin1 and Caveolin-1, but only after adrenergic stimulation. This was confirmed by immunoprecipitation from Caveolin-1 containing membrane fractions. To assess the functional consequences of this transient interaction, we generated an inducible VSMC-specific Caveolin-1 KO mouse. Deletion of Caveolin-1 from VSMC of resistance arteries significantly blunted adrenergic induced ATP release and vasoconstriction. Using radiotelemetry to assess changes in systemic blood pressure, we observed a significant reduction in mean arterial pressure only after Caveolin-1 deletion, which occurred at night when murine sympathetic activity is high. These findings closely recapitulate our published observations in VSMC specific Pannexin1 knock out mice. Moreover, with VSMC Caveolin-1 deletion, we demonstrate resistance to acute blood pressure lowering following treatment with the Pannexin1 inhibitory peptide PanX. Thus, the localization of Pannexin1 to Caveolin-1 at the plasma membrane could facilitate AR-mediated ATP release and vasoconstriction necessary for blood pressure regulation. O-2 MODULATION OF CX37-CONTAINING GAP JUNCTIONS BY SHP-2 AND ITS FUNCTIONAL CONSEQUENCE FOR MYOENDOTHELIAL COMMUNICATION Petra Kameritsch1, Kristin Pogoda1, Hanna Mannell1, Ulrich Pohl1 1Walter Brendel Centre, Ludwig-Maximilians University, Germany We have shown that nitric oxide (NO) inhibits communication via connexin 37 (Cx37)-containing gap junctions (GJ). Here we investigated the NO-induced post-translational modification in the C- terminus of Cx37 and its functional consequence for myoendothelial calcium signal transfer and endothelium dependent dilation of isolated small resistance arteries. Calcium (Fura-2) was increased by mechanical stimulation of HeLa cells expressing Cx37 variants, HUVEC transfected with SHP-2 variants or single ECs of isolated mouse resistance arteries and the signal propagation was analysed respectively (n=8-29). The phosphorylation status of a synthetic Cx37-peptide (AA 324-333) with a phosphorylated tyrosine (“P-Tyr”) equivalent to Tyr 332 was measured after incubation with EC or HeLa cell lysates (MALDI-TOF, n=5-15). The effect of NO-treatment on ACh-induced endothelial calcium increases and dilator responses of isolated vessels (pretreated with 10 µM ODQ to inhibit cGMP) were studied. (n=4-6). NO significantly reduced the gap junction dependent calcium signal propagation only in cells in which Tyr 332 of Cx37 was present. Impairment of the tyrosine phosphatase SHP-2 by dominant negative mutants or pharmacologic inhibitors abolished the NO effect. Lysates of control cells but not of NO-treated cells or cells lacking functional SHP-2 de-phosphorylated the synthetic peptide (con: 1.03±0.18 Tyr/P-Tyr; NO: 0.55±0.05 Tyr/P-Tyr; SHP-2-deficient con: 0.49±0.12 Tyr/P-Tyr; SHP-2-deficient NO: 0.43±0.08 Tyr/P-Tyr). NO-treatment increased the calcium signal propagation within the endothelial layer of isolated vessels, whereas it significantly decreased the signal propagation from endothelial into smooth muscle cells. The increased endothelial calcium went along with a significant leftward shift of the ACh dose effect relation. We conclude that NO, inhibits Cx37 dependent calcium transfer by increasing its phosphorylation due to a blockade of SHP-2 activity. As a result, in myoendothelial gap junctions of intact vessels, the endothelial calcium loss into smooth muscle is reduced leading to an enhanced, probably EDH-mediated, dilator response of arterial resistance vessels. O-3 TARGETING CONNEXIN40 REDUCES ANGIOGENESIS IN THE DEVELOPING MOUSE RETINA Florian Alonso2, Florent Allagnat1, Lauriane Hamard1, Loïc Le Gal1, Paolo Meda1, Denise Nardelli Haefliger1, Elisabeth Génot2, Jacques-Antoine Haefliger1 1CHUV Hospital, Switzerland, 2Cardio-Thoracic Research Center of Bordeaux, France Connexin40 (Cx40) forms intercellular channels that coordinate the electrical conduction in the heart and the vasomotor tone in large vessels. We previously documented that altering Cx40 expression or function in endothelial cells (EC) impairs their ability to vascularize tumors. Using Cx40 deficient mice (Cx40-/-) and the neovascularization model of the mouse post-natal retina, we found that Cx40 contributes to physiological angiogenesis. Genetic deletion of Cx40 leads to a reduction in vascular growth and capillary density in the vascular network of the developing mouse retina. At the angiogenic front, vessel sprouting is reduced, and the mural cells recruited along the sprouts display an altered phenotype. These alterations can be attributed to disturbed EC functions as selective re-expression of Cx40 in these cells restores normal angiogenesis. In vitro, targeting Cx40 in microvascular EC, by silencing its expression or by blocking gap junction channels, decreases their proliferation, modifies their secretion of PDGF and promotes the chemoattraction of mural cells. In vivo, an intravitreal injection of a Cx40 inhibitory peptide phenocopies the loss of Cx40 in the retinal vasculature of wild type mice. Collectively, our data show that endothelial Cx40 regulates vessel growth and maturation in the developing retina and may represent a novel therapeutic target for treating pathological ocular angiogenesis. O-4 3D ELECTRON MICROSCOPY COMBINED WITH MULTIPLEX IMMUNOGOLD LABELING TO LOCALIZE PHOSPHORYLATED FORMS OF CONNEXIN 43 IN OVARIAN GRANULOSA CELLS Rachael Norris1, Valentina Baena1, Joell Solan2, Paul Lampe2, Mark Terasaki1 1UConn Health, United States, 2Fred Hutchinson Cancer Research Center, United States While correlative links between Cx43 phosphorylation and gap junction internalization are well recognized, specifically phosphorylated forms of Cx43 have not been localized at the resolution needed to discern individual gap junctions from invaginating gap junctions or from connexosomes near the plasma membrane. To do this, we have established a method for immunogold labeling of serial ultrathin sections collected on tape and imaged with scanning electron microscopy. The ovarian follicle is well suited to studying Cx43 phosphorylation and internalization because gap junctions that connect granulosa cells are modified when meiotic resumption is stimulated with luteinizing hormone (LH). Specifically, Cx43 phosphorylation increases on MAP kinase sites S262 and S279/282, gap junction permeability transiently decreases, and the number of connexosomes found in granulosa cells is increased. Using specific Cx43 antibodies against phosphorylated S262, S279/282, S368 and S373, we labeled gap junctions and connexosomes in ovarian follicles before and during meiotic resumption. Before LH stimulation, we find that Cx43 is phosphorylated on S368 in all gap junctions and connexosomes examined while phosphorylation on S262 only occurs in about half of the connexosomes and in none of the gap junctions. Thirty minutes after LH stimulation, we see decreased S368 phosphorylation of Cx43 in both gap junctions and connexosomes. Conversely, phosphorylation on S262 is present in most connexosomes and in some gap junctions. Since gap junctions and connexosomes span several ultrathin sections, we labeled individual structures with two or more phosphospecific antibodies by applying different antibodies to separate serial sections. With this technique, we find that Cx43 in individual connexosomes can be phosphorylated on all the sites we examined. Now that we can detect individual gap junctions or connexosomes and label them with multiple antibodies, we will advance our understanding of the spatiotemporal phosphorylation events that regulate gap junction turnover. O-5 INTRAMOLECULAR SIGNALLING IN A CARDIAC CONNEXIN: ROLE OF CYTOPLASMIC DOMAIN DIMERIZATION Andrew Trease1, Juan Capuccino2, Jorge Contreras2,
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