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Knockdown of Add3 impairs the myogenic response of renal afferent arterioles and middle cerebral arteries.

ABSTRACT: We have reported that the myogenic response of the renal afferent arteriole (Af-art) and middle cerebral artery (MCA) and autoregulation of renal and cerebral blood flow are impaired in Fawn-Hooded Hypertensive (FHH) rats. Transfer of a region of chromosome 1 containing γ-adducin (Add3) from the Brown Norway rat rescued the vascular dysfunction and the development of renal disease. To examine whether Add3 is a viable candidate gene altering renal and cerebral hemodynamics in FHH rats, we knocked down the expression of Add3 in rat Af-arts and MCAs cultured for 36-h using a 27-mer Dicer-substrate short interfering RNA (DsiRNA). Control Af-arts constricted by 10 ± 1% in response to an elevation in pressure from 60 to 120 mmHg but dilated by 4 ± 3% when treated with Add3 DsiRNA. Add3 DsiRNA had no effect on the vasoconstrictor response of the Af-art to norepinephrine (10-7 M). Add3 DsiRNA had a similar effect on the attenuation of the myogenic response in the MCA. Peak potassium currents were threefold higher in smooth muscle cells isolated from Af-arts or MCAs transfected with Add3 DsiRNA than in nontransfected cells isolated from the same vessels. This is the first study demonstrating that Add3 plays a role in the regulation of potassium channel function and vascular reactivity. It supports the hypothesis that sequence variants in Add3, which we previously identified in FHH rats, may play a causal role in the impaired myogenic response and autoregulation in the renal and cerebral circulation.

SUBMITTER: Fan F

PROVIDER: S-EPMC5495887 | biostudies-other | 2017 Jun

REPOSITORIES: biostudies-other

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Knockdown of Add3 impairs the myogenic response of renal afferent arterioles and middle cerebral arteries.

Fan Fan F Pabbidi Mallikarjuna R MR Ge Ying Y Li Longyang L Wang Shaoxun S Mims Paige N PN Roman Richard J RJ

American journal of physiology. Renal physiology 20161207 6

We have reported that the myogenic response of the renal afferent arteriole (Af-art) and middle cerebral artery (MCA) and autoregulation of renal and cerebral blood flow are impaired in Fawn-Hooded Hypertensive (FHH) rats. Transfer of a region of chromosome 1 containing γ-adducin (Add3) from the Brown Norway rat rescued the vascular dysfunction and the development of renal disease. To examine whether Add3 is a viable candidate gene altering renal and cerebral hemodynamics in FHH rats, we knocked ...[more]

PMID: 27927653

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Project description:Pressure-induced arterial depolarization and constriction (the myogenic response) is a smooth muscle cell (myocyte)-specific mechanism that controls regional organ blood flow and systemic blood pressure. Several different nonselective cation channels contribute to pressure-induced depolarization, but signaling mechanisms involved are unclear. Similarly uncertain is the contribution of anion channels to the myogenic response and physiological functions and mechanisms of regulation of recently discovered transmembrane 16A (TMEM16A), also termed Anoctamin 1, chloride (Cl(-)) channels in arterial myocytes.To investigate the hypothesis that myocyte TMEM16A channels control membrane potential and contractility and contribute to the myogenic response in cerebral arteries.Cell swelling induced by hyposmotic bath solution stimulated Cl(-) currents in arterial myocytes that were blocked by TMEM16A channel inhibitory antibodies, RNAi-mediated selective TMEM16A channel knockdown, removal of extracellular calcium (Ca(2+)), replacement of intracellular EGTA with BAPTA, a fast Ca(2+) chelator, and Gd(3+) and SKF-96365, nonselective cation channel blockers. In contrast, nimodipine, a voltage-dependent Ca(2+) channel inhibitor, or thapsigargin, which depletes intracellular Ca(2+) stores, did not alter swelling-activated TMEM16A currents. Pressure-induced (-40 mm Hg) membrane stretch activated ion channels in arterial myocyte cell-attached patches that were inhibited by TMEM16A antibodies and were of similar amplitude to recombinant TMEM16A channels. TMEM16A knockdown reduced intravascular pressure-induced depolarization and vasoconstriction but did not alter depolarization-induced (60 mmol/L K(+)) vasoconstriction.Membrane stretch activates arterial myocyte TMEM16A channels, leading to membrane depolarization and vasoconstriction. Data also provide a mechanism by which a local Ca(2+) signal generated by nonselective cation channels stimulates TMEM16A channels to induce myogenic constriction.

Project description:New findingsWhat is the central question of this study? DAPK3 contributes to the Ca2+ -sensitization of vascular smooth muscle contraction: does this protein kinase participate in the myogenic response of cerebral arteries? What is the main finding and its importance? Small molecule inhibitors of DAPK3 effectively block the myogenic responses of cerebral arteries. HS38-dependent changes to vessel constriction occur independent of LC20 phosphorylation, and therefore DAPK3 appears to operate via the actin cytoskeleton. A role for DAPK3 in the myogenic response was not previously reported, and the results support a potential new therapeutic target in the cerebrovascular system.AbstractThe vascular smooth muscle (VSM) of resistance blood vessels is a target of intrinsic autoregulatory responses to increased intraluminal pressure, the myogenic response. In the brain, the myogenic reactivity of cerebral arteries is critical to homeostatic blood flow regulation. Here we provide the first evidence to link the death-associated protein kinase 3 (DAPK3) to the myogenic response of rat and human cerebral arteries. DAPK3 is a Ser/Thr kinase involved in Ca2+ -sensitization mechanisms of smooth muscle contraction. Ex vivo administration of a specific DAPK3 inhibitor (i.e., HS38) could attenuate vessel constrictions invoked by serotonin as well as intraluminal pressure elevation. The HS38-dependent dilatation was not associated with any change in myosin light chain (LC20) phosphorylation. The results suggest that DAPK3 does not regulate Ca2+ sensitization pathways during the myogenic response of cerebral vessels but rather operates to control the actin cytoskeleton. A slow return of myogenic tone was observed during the sustained ex vivo exposure of cerebral arteries to HS38. Recovery of tone was associated with greater LC20 phosphorylation that suggests intrinsic signalling compensation in response to attenuation of DAPK3 activity. Additional experiments with VSM cells revealed HS38- and siDAPK-dependent effects on the actin cytoskeleton and focal adhesion kinase phosphorylation status. The translational importance of DAPK3 to the human cerebral vasculature was noted, with robust expression of the protein kinase and significant HS38-dependent attenuation of myogenic reactivity found for human pial vessels.

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Knockdown of Add3 impairs the myogenic response of renal afferent arterioles and middle cerebral arteries.

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Knockdown of Add3 impairs the myogenic response of renal afferent arterioles and middle cerebral arteries.

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