Microtubules can regulate GPCR (G protein-coupled receptor) signaling in various cell types. In vascular smooth muscle, activation of the β-adrenoceptor leads to production of cAMP to mediate a vasorelaxation. Little is known about the role of microtubules in smooth muscle, and given the importance of this pathway in vascular smooth muscle cells, we investigated the role of microtubule stability on β-adrenoceptor signaling in rat renal and mesenteric arteries. In isometric tension experiments, incubation with the microtubule inhibitors colchicine and nocodazole enhanced isoprenaline-mediated relaxations of renal and mesenteric arteries that the microtubule stabilizer, paclitaxel, prevented. Sharp microelectrode experiments showed that colchicine treatment caused increased hyperpolarization of mesenteric artery segments in response to isoprenaline. Application of the Kv7 channel blocker, XE991, attenuated the effect of colchicine on isoprenaline relaxations, whereas iberiotoxin-a BKCa channel blocker-had no effect. In addition, colchicine improved the relaxations to the Kv7.2 to 7.5 activator, S-1, in both renal and mesenteric artery segments compared with dimethyl sulfoxide incubation. We determined that increased mesenteric artery myocytes treated with colchicine showed increased Kv7.4 membrane expression, but Western blot analysis showed no change in total Kv7.4 protein. This study is the first to show microtubule disruption improves the β-adrenoceptor-mediated relaxations of mesenteric and renal arteries and determine this enhancement to be because of increased membrane expression of the Kv7 voltage-gated potassium channels.

These data show that 4-AP is able to relax NA-preconstricted rat mesenteric arteries by enhancing the activity of K 7.4 and BK channels and attenuating NA-mediated signalling.

Aim:This study aimed to assess intracellular Ca 2+ dynamics in nerve cells and Schwann cells in isolated rat resistance arteries and determine how these dynamics modify noradrenaline release from the nerves and consequent force development. Methods: Ca 2+ in nerves was assessed with confocal imaging, noradrenaline release with amperometry and artery tone with wire myography. Ca 2+ in axons was assessed after loading with Oregon Green 488 BAPTA-1 dextran. In other experiments, arteries were incubated with Calcium Green-1-AM which loads both axons and Schwann cells. Results: Schwann cells but not axons responded with a Ca 2+ increase to ATP.Electrical field stimulation of nerves caused a frequency-dependent increase in varicose [Ca 2+ ] ([Ca 2+ ] v ). ω-conotoxin-GVIA (100 nmol/L) reduced the [Ca 2+ ] v transient to 2 and 16 Hz by 60% and 27%, respectively; in contrast ω-conotoxin GVIA inhibited more than 80% of the noradrenaline release and force development at 2 and 16 Hz. The K V channel blocker, 4-aminopyridine (10 µmol/L), increased [Ca 2+ ] v , noradrenaline release and force development both in the absence and presence of ωconotoxin-GVIA. Yohimbine (1 µmol/L) increased both [Ca 2+ ] v and noradrenaline release but reduced force development. Acetylcholine (10 µmol/L) caused atropinesensitive inhibition of [Ca 2+ ] v , noradrenaline release and force. In the presence of ω-conotoxin-GVIA, acetylcholine caused a further inhibition of all parameters. Conclusion: Modification of [Ca 2+ ] in arterial sympathetic axons and Schwann cells was assessed separately. K V 3.1 channels may be important regulators of [Ca 2+ ] v , noradrenaline release and force development. Presynaptic adrenoceptor and muscarinic receptor activation modify transmitter release through modification of [Ca 2+ ] v . K E Y W O R D S amperometry, confocal imaging, neurotransmission, prejunctional modulation, small arteries, sympathetic 2 of 11 | HANSEN Et Al.

We investigated the acute effects of glucagon-like peptide-1 (GLP-1), GLP-1(1-36), and GLP-1(7-36) on vascular endothelial growth factor-A (VEGFA)-induced endothelium-dependent signaling and vasodilation. Our hypothesis was that GLP-1 released from intestinal l-cells modulates processes related to PLCγ activation, Src, and endothelial NOS (eNOS) signaling, thereby controlling endothelial vessel tone. By using RT-PCR analysis, we found mRNA for the GLP-1 receptor (GLP-1R) in human dermal microvascular endothelial cells (HDMEC), human retinal microvascular endothelial cells, and rat arteries. In isolated rat mesenteric resistance arteries precontracted with the thromboxane analog U46619 to 80-90% of maximum contraction, VEGFA (25 ng/ml) caused a small and gradual relaxation (28.9 ± 3.9%). Pretreatment of arteries with either GLP-1(1-36) (500 nM) or GLP-1(7-36) (1 nM) abolished the VEGFA-induced relaxation. VEGFA-induced relaxations were also inhibited in endothelial-denuded arteries and in arteries pretreated with the nitric oxide synthase (NOS) inhibitor, Nω-nitro-l-arginine methyl ester (100 μM). In vivo studies on male Wistar rats also revealed that GLP-1(7-36) inhibited VEGFA-induced vasodilation of the same arteries. In isolated endothelial cells, GLP-1(1-36) and GLP-1(7-36) caused a reduction in VEGFA-induced phosphorylation of PLCγ. Ca imaging of endothelial cells and rat mesenteric resistance arteries using fura-2, revealed that both GLP-1 analogs caused a reduction in VEGFA-induced Ca signaling. GLP-1(1-36) also reduced VEGFA-induced eNOS phosphorylation in HDMEC. In conclusion, GLP-1 reduced relaxation induced by VEGFA in resistance arteries by inhibiting VEGFR2-mediated Ca signaling and endothelial NO synthesis. GLP-1, on its own, also induced phosphorylation of Src and ERK1/2 that can lead to proliferation and is implicated in vessel permeability.

*Contributed equally. BACKGROUND AND PURPOSEPDE1, a subfamily of cyclic nucleotide PDEs consisting of three isoforms, PDE1A, PDE1B and PDE1C, has been implicated in the regulation of vascular tone. The PDE1 isoform(s) responsible for tone regulation is unknown. This study used isoform-preferring PDE1 inhibitors, Lu AF58027, Lu AF64196, Lu AF66896 and Lu AF67897, to investigate the relative contribution of PDE1 isoforms to regulation of vascular tone. EXPERIMENTAL APPROACHIn rat mesenteric arteries, expression and localization of Pde1 isoforms were determined by quantitative PCR and in situ hybridization, and physiological impact of PDE1 inhibition was evaluated by isometric tension recordings. KEY RESULTSIn rat mesenteric arteries, Pde1a mRNA expression was higher than Pde1b and Pde1c. In situ hybridization revealed localization of Pde1a to vascular smooth muscle cells (VSMCs) and only minor appearance of Pde1b and Pde1c. The potency of the PDE1 inhibitors at eliciting relaxation showed excellent correlation with their potency at inhibiting PDE1A. Thus, Lu AF58027 was the most potent at inhibiting PDE1A and was also the most potent at eliciting relaxation in mesenteric arteries. Inhibition of NOS with L-NAME, soluble GC with ODQ or PKG with Rp-8-Br-PET-cGMP all attenuated the inhibitory effect of PDE1 on relaxation, whereas PKA inhibition with H89 had no effect. CONCLUSIONS AND IMPLICATIONSPde1a is the dominant PDE1 isoform present in VSMCs, and relaxation mediated by PDE1A inhibition is predominantly driven by enhanced cGMP signalling. These results imply that isoform-selective PDE1 inhibitors are powerful investigative tools allowing examination of physiological and pathological roles of PDE1 isoforms. AbbreviationsCCh, carbachol; H89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; ODQ, 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one; Rp-8, Rp-8-Br-PET-cGMPS; U46619, 9 α-epoxymethanoPGF 2α ; VSMCs, vascular smooth muscle cells

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Background: Development of aortic regurgitation/insufficiency has been observed in patients implanted with continuous flow axial left ventricular assist devices. The aim of this study was to examine the development of aortic regurgitation/insufficiency in patients implanted with continuous flow centrifugal left ventricular assist devices.Methods: A retrospective analysis of patients implanted with the VentraAssist continuous flow LVAD (n = 33) and Heartware (n = 23) was performed. Echocardiographic data from pre-implant, and at last follow-up was analysed for the development of aortic regurgitation (AR). Left ventricular dimensions were recorded. AR was scored from 0 = nil; 2 = mild; 4 = moderate; 6 = severe.Results: Four of the 56 patients had grade 2 AR prior to device implant (7.1%). In patients with long term follow-up data (n = 23, mean days since implant 367 ± 25), LV enddiastolic dimension (LVEDD) decreased from 77.2 ± 12.3 to 70.7 ± 12.2 mm (p = 0.001). AR increased from a mean grade of 0.12 to a mean grade of 0.70. Only one patient with documented AR at day 360 had a partially open aortic valve.Conclusion: A proportion of patients implanted with continuous flow centrifugal LVADs develop aortic insufficiency/regurgitation with time. This is likely to be due to the closed aortic valve being exposed to continuously increased aortic root back-pressure. Despite the development of aortic regurgitation, an improvement in LVEDD with mechanical unloading is noted.