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Key points Depletion of Ca2+ stores activates store-operated channels (SOCs), which mediate Ca2+ entry pathways that regulate cellular processes such as contraction, proliferation and gene expression., In vascular smooth muscle cells (VSMCs), stimulation of SOCs composed of canonical transient receptor potential channel 1 (TRPC1) proteins requires G protein α q subunit (Gαq)/phospholipase C (PLC)β1/protein kinase C (PKC) activity. We studied the role of stromal interaction molecule 1 (STIM1) in coupling store depletion to this activation pathway using patch clamp recording, GFP-PLCδ1-PH imaging and co-localization techniques., Store-operated TRPC1 channel and PLCβ1 activities were inhibited by STIM1 short hairpin RNA (shRNA) and absent in TRPC1−/− cells, and store-operated PKC phosphorylation of TRPC1 was inhibited by STIM1 shRNA. Store depletion induced interactions between STIM1 and TRPC1, Gαq and PLCβ1, which required STIM1 and TRPC1. Similar effects were produced with noradrenaline., These findings identify a new activation mechanism of TRPC1-based SOCs in VSMCs, and a novel role for STIM1, where store-operated STIM1-TRPC1 interactions stimulate Gαq/PLCβ1/PKC activity to induce channel gating., Abstract In vascular smooth muscle cells (VSMCs), stimulation of canonical transient receptor potential channel 1 (TRPC1) protein-based store-operated channels (SOCs) mediates Ca2+ entry pathways that regulate contractility, proliferation and migration. It is therefore important to understand how these channels are activated. Studies have shown that stimulation of TRPC1-based SOCs requires G protein α q subunit (Gαq)/phospholipase C (PLC)β1 activities and protein kinase C (PKC) phosphorylation, although it is unclear how store depletion stimulates this gating pathway. The present study examines this issue by focusing on the role of stromal interaction molecule 1 (STIM1), an endo/sarcoplasmic reticulum Ca2+ sensor. Store-operated TRPC1 channel activity was inhibited by TRPC1 and STIM1 antibodies and STIM1 short hairpin RNA (shRNA) in wild-type VSMCs, and was absent in TRPC1−/− VSMCs. Store-operated PKC phosphorylation of TRPC1 was reduced by knockdown of STIM1. Moreover, store-operated PLCβ1 activity measured with the fluorescent phosphatidylinositol 4,5-bisphosphate/inositol 1,4,5-trisphosphate biosensor GFP-PLCδ1-PH was reduced by STIM1 shRNA and absent in TRPC1−/− cells. Immunocytochemistry, co-immunoprecipitation and proximity ligation assays revealed that store depletion activated STIM1 translocation from within the cell to the plasma membrane (PM) where it formed STIM1-TRPC1 complexes, which then associated with Gαq and PLCβ1. Noradrenaline also evoked TRPC1 channel activity and associations between TRPC1, STIM1, Gαq and PLCβ1, which were inhibited by STIM1 knockdown. Effects of N-terminal and C-terminal STIM1 antibodies on TRPC1-based SOCs and STIM1 staining suggest that channel activation may involve insertion of STIM1 into the PM. The findings of the present study identify a new activation mechanism of TRPC1-based SOCs in VSMCs, and a novel role for STIM1, in which store-operated STIM1-TRPC1 interactions stimulate PLCβ1 activity to induce PKC phosphorylation of TRPC1 and channel gating. [ABSTRACT FROM AUTHOR]

Aim Rats with adenine-induced chronic renal failure (A- CRF) develop a reduced rate of relaxation of the thoracic aorta. The aim of this study was to elucidate the mechanisms underlying this abnormality. Methods Male Sprague Dawley rats received either chow containing adenine or were pair-fed with normal chow (controls). After 8-14 weeks, arterial function was analysed ex vivo using wire myography and the expression of proteins involved in vascular smooth muscle excitation-contraction coupling in the thoracic aorta was analysed. Results The rate of relaxation following washout of KCl was reduced in A- CRF rats vs. controls in the thoracic aorta ( P < 0.01), abdominal aorta ( P < 0.05), and common carotid artery ( P < 0.05), but not in the common femoral artery. Relaxation rates of thoracic aortas increased ( P < 0.01), but were not normalized, in response to washout of KCl with Ca2+-free buffer. Microarray and qRT- PCR analyses of genes involved in excitation-contraction coupling identified 10 genes, which showed significantly altered expression in A- CRF thoracic aortas. At the protein level, the α2 subunit of the Na,K- ATPase ( P < 0.001) and SERCA2 ( P < 0.05) was significantly downregulated, whereas stromal interaction molecule 1 and calsequestrin-1 and calsequestrin-2 were significantly upregulated ( P < 0.05). Conclusions Rats with A- CRF show a marked alteration in relaxation of larger conduit arteries localized proximal to the common femoral artery. This abnormality may be caused by reduced cytosolic Ca2+ clearance in vascular smooth muscle cells secondary to dysregulation of proteins crucially involved in this process. [ABSTRACT FROM AUTHOR]

Key points Cyclic stretch is known to alter intracellular pathways involved in vessel tone regulation., We developed a novel set-up that allows straightforward characterization of the biomechanical properties of the mouse aorta while stretched at a physiological heart rate (600 beats min-1)., Active vessel tone was shown to have surprisingly large effects on isobaric stiffness., The effect of structural vessel wall alterations was confirmed using a genetic mouse model., This set-up will contribute to a better understanding of how active vessel wall components and mechanical stimuli such as stretch frequency and amplitude regulate aortic mechanics., Abstract Cyclic stretch is a major contributor to vascular function. However, isolated mouse aortas are frequently studied at low stretch frequency or even in isometric conditions. Pacing experiments in rodents and humans show that arterial compliance is stretch frequency dependent. The Rodent Oscillatory Tension Set-up to study Arterial Compliance is an in-house developed organ bath set-up that clamps aortic segments to imposed preloads at physiological rates up to 600 beats min-1. The technique enables us to derive pressure-diameter loops and assess biomechanical properties of the segment. To validate the applicability of this set-up we aimed to confirm the effects of distension pressure and vascular smooth muscle tone on arterial stiffness. At physiological stretch frequency (10 Hz), the Peterson modulus ( EP; 293 (10) mmHg) for wild-type mouse aorta increased 22% upon a rise in pressure from 80-120 mmHg to 100-140 mmHg, while, at normal pressure, EP increased 80% upon maximal contraction of the vascular smooth muscle cells. We further validated the method using a mouse model with a mutation in the fibrillin-1 gene and an endothelial nitric oxide synthase knock-out model. Both models are known to have increased arterial stiffness, and this was confirmed using the set-up. To our knowledge, this is the first set-up that facilitates the study of biomechanical properties of mouse aortic segments at physiological stretch frequency and pressure. We believe that this set-up can contribute to a better understanding of how cyclic stretch frequency, amplitude and active vessel wall components influence arterial stiffening. [ABSTRACT FROM AUTHOR]

Background and Purpose: We investigated the hypothesis that elevated glucose increases contractile responses in vascular smooth muscle and that this enhanced constriction occurs due to the glucose-induced PKC-dependent inhibition of voltage-gated potassium channels.Experimental Approach: Patch-clamp electrophysiology in rat isolated mesenteric arterial myocytes was performed to investigate the glucose-induced inhibition of voltage-gated potassium (Kv ) current. To determine the effects of glucose in whole vessel, wire myography was performed in rat mesenteric, porcine coronary and human internal mammary arteries.Key Results: Glucose-induced inhibition of Kv was PKC-dependent and could be pharmacologically dissected using PKC isoenzyme-specific inhibitors to reveal a PKCβ-dependent component of Kv inhibition dominating between 0 and 10 mM glucose with an additional PKCα-dependent component becoming evident at concentrations greater than 10 mM. These findings were supported using wire myography in all artery types used, where contractile responses to vessel depolarization and vasoconstrictors were enhanced by increasing bathing glucose concentration, again with evidence for distinct and complementary PKCα/PKCβ-mediated components.Conclusions and Implications: Our results provide compelling evidence that glucose-induced PKCα/PKCβ-mediated inhibition of Kv current in vascular smooth muscle causes an enhanced constrictor response. Inhibition of Kv current causes a significant depolarization of vascular myocytes leading to marked vasoconstriction. The PKC dependence of this enhanced constrictor response may present a potential therapeutic target for improving microvascular perfusion following percutaneous coronary intervention after myocardial infarction in hyperglycaemic patients. [ABSTRACT FROM AUTHOR]

Background and Purpose Calcium-activated chloride channels ( CaCCs) are key depolarizing mechanisms that have an important role in vascular smooth muscle contraction. Here, we investigated whether these channels are regulated by phosphatidylinositol (4,5) bisphosphate [ P(4,5) P2], a known regulator of various ion channels. Experimental Approach Calcium-activated Cl− currents ( IClCa) were recorded by patch clamp electrophysiology of rat isolated pulmonary artery smooth muscle cells. TMEM16A protein-phosphoinositide interaction was studied by co-immunoprecipitation and phosphoinositide binding arrays on protein lysates from whole pulmonary arteries and HEK293 cells overexpressing TMEM16A, the molecular correlate. Key Results PI(4,5) P2 and other phospholipids were shown to bind directly to TMEM16A isolated from whole pulmonary artery ( PA) and TMEM16A-eGFP expressed in HEK293 cells. Agents that reduced PI(4,5) P2 levels through different routes [PL C activation, PI4K inhibition, PI(4,5) P2 scavenging and absorption] all increased IClCa evoked by solutions containing clamped-free [ Ca2+], whereas enrichment of activating solutions with PI(4,5) P2 inhibited IClca in PA smooth muscle cells with approximately 50% reduction at 1 μM. Conclusions and Implications These data are the first to show a negative regulation of TMEM16A-encoded CaCCs by PI(4,5) P2 and propose that control of PI(4,5) P2 levels is a key determinant of arterial physiology. [ABSTRACT FROM AUTHOR]

Vascular smooth muscle contraction and the myogenic response regulate blood flow in the resistance vascular and contribute to systemic blood pressure. Three pathways are currently known to contribute to the development of the myogenic response: (i) Ca2+-dependent phosphorylation of LC20; (ii) Ca2+ sensitization through inhibition of myosin phosphatase; and (iii) cortical actin polymerization. A number of regulatory smooth muscle proteins are integrated with these pathways to fine tune the response and facilitate adaptations to vascular (patho)physiologies. Of particular interest is the SMTN family of proteins, consisting of SMTN-A, SMTN-B, and the SMTN-like protein, SMTNL1. The SMTN-B and SMTNL1 proteins are both implicated in regulating smooth muscle contractility and contributing to vascular adaptations associated with hypertension, pregnancy, and exercise training. In the case of SMTNL1, the protein plays multiple roles in regulating contraction through functional interactions with contractile regulators as well as transcriptional control of the contractile phenotype and Ca2+-sensitizing capacity. For the first time, preliminary results suggest SMTNL1 is involved in the myogenic response of the cerebral resistance vasculature. In this regard, global SMTNL1 deletion is associated with greater myogenic reactivity of cerebral arterioles, although the precise mechanism accounting for this finding remains to be defined. [ABSTRACT FROM AUTHOR]