Arterial Myocytes Research Articles

Adenylyl cyclase 5-generated cAMP controls cerebral vascular reactivity during diabetic hyperglycemia.

Elevated blood glucose (hyperglycemia) is a hallmark metabolic abnormality in diabetes. Hyperglycemia is associated with protein kinase A (PKA)-mediated stimulation of L-type Ca2+ channels in arterial myocytes resulting in increased vasoconstriction. However, the mechanisms by which glucose activates PKA remain unclear. Here, we showed that elevating extracellular glucose stimulates cAMP production in arterial myocytes, and that this was specifically dependent on adenylyl cyclase 5 (AC5) activity. Super-resolution imaging suggested nanometer proximity between subpopulations of AC5 and the L-type Ca2+ channel pore-forming subunit CaV1.2. In vitro, in silico, ex vivo and in vivo experiments revealed that this close association is critical for stimulation of L-type Ca2+ channels in arterial myocytes and increased myogenic tone upon acute hyperglycemia. This pathway supported the increase in L-type Ca2+ channel activity and myogenic tone in two animal models of diabetes. Our collective findings demonstrate a unique role for AC5 in PKA-dependent modulation of L-type Ca2+ channel activity and vascular reactivity during acute hyperglycemia and diabetes.

Membrane Stretch-Induced Activation of a TRPM4-Like Nonselective Cation Channel in Cerebral Artery Myocytes

Membrane Stretch-Induced Activation of a TRPM4-Like Nonselective Cation Channel in Cerebral Artery Myocytes

The cell-wide web coordinates cellular processes by directing site-specific Ca2+ flux across cytoplasmic nanocourses

Ca2+ coordinates diverse cellular processes, yet how function-specific signals arise is enigmatic. We describe a cell-wide network of distinct cytoplasmic nanocourses with the nucleus at its centre, demarcated by sarcoplasmic reticulum (SR) junctions (≤400 nm across) that restrict Ca2+ diffusion and by nanocourse-specific Ca2+-pumps that facilitate signal segregation. Ryanodine receptor subtype 1 (RyR1) supports relaxation of arterial myocytes by unloading Ca2+ into peripheral nanocourses delimited by plasmalemma-SR junctions, fed by sarco/endoplasmic reticulum Ca2+ ATPase 2b (SERCA2b). Conversely, stimulus-specified increases in Ca2+ flux through RyR2/3 clusters selects for rapid propagation of Ca2+ signals throughout deeper extraperinuclear nanocourses and thus myocyte contraction. Nuclear envelope invaginations incorporating SERCA1 in their outer nuclear membranes demarcate further diverse networks of cytoplasmic nanocourses that receive Ca2+ signals through discrete RyR1 clusters, impacting gene expression through epigenetic marks segregated by their associated invaginations. Critically, this circuit is not hardwired and remodels for different outputs during cell proliferation.

The Selective Rat Toxicant Norbormide Blocks KATP Channels in Smooth Muscle Cells But Not in Insulin-Secreting Cells.

Norbormide is a toxicant selective for rats to which it induces a widespread vasoconstriction. In a recent paper, we hypothesized a role of ATP-sensitive potassium (KATP) channels in norbormide-induced vasoconstriction. The current study was undertaken to verify this hypothesis by comparing the effects of norbormide with those of glibenclamide, a known KATP channel blocker. The whole-cell patch-clamp method was used to record KATP currents in myocytes freshly isolated from the rat and mouse caudal artery and from the rat gastric fundus, as well as in insulin-secreting pancreatic beta cells (INS-1 cells). Smooth muscle contractile function was assessed on either rat caudal artery rings or gastric fundus strips. Molecular modeling and docking simulation to KATP channel proteins were investigated in silico. Both norbormide (a racemic mixture of endo and exo isomers) and glibenclamide inhibited KATP currents in rat and mouse caudal artery myocytes, as well as in gastric fundus smooth muscle cells. In rat INS-1 cells, only glibenclamide blocked KATP channels, whereas norbormide was ineffective. The inhibitory effect of norbormide in rat caudal artery myocytes was not stereo-specific as both the endo isomers (active as vasoconstrictor) and the exo isomers (inactive as vasoconstrictor) had similar inhibitory activity. In rat caudal artery rings, norbormide-induced contraction was partially reverted by the KATP channel opener pinacidil. Computational approaches indicated the SUR subunit of KATP channels as the binding site for norbormide. KATP channel inhibition may play a role in norbormide-induced vasoconstriction, but does not explain the species selectivity, tissue selectivity, and stereoselectivity of its constricting activity. The lack of effect in INS-1 cells suggests a potential selectivity of norbormide for smooth muscle KATP channels.

Increased TRPM4 Activity in Cerebral Artery Myocytes Contributes to Cerebral Blood Flow Reduction After Subarachnoid Hemorrhage in Rats.

Cerebral blood flow (CBF) reduction underlies unfavorable outcomes after subarachnoid hemorrhage (SAH). Transient receptor potential melastatin-4 (TRPM4) has a pivotal role in cerebral artery myogenic tone maintenance and CBF regulation under physiological conditions. However, the role of TRPM4 in CBF reduction after SAH is unclear. In this study, we aimed at testing whether TRPM4 would contribute to CBF reduction after SAH in vivo and determining underlying mechanisms. Rat SAH model was established by stereotaxic injection of autologous nonheparinized arterial blood at the suprasellar cistern. A TRPM4 blocker, 9-phenanthrol (9-Phe), was infused through an intraventricular catheter connected to a programmed subcutaneous pump to evaluate the contribution of TRPM4 to SAH outcomes. TRPM4 expression and translocation in cerebral artery myocytes were detected by immunoblotting. Macroscopic currents in cerebral artery myocytes were determined by whole-cell patch clamp. Myogenic tone of cerebral arteries was studied by pressurized myography. Cortical and global CBFs were measured via laser Doppler flowmetry and fluorescent microspheres, respectively. After SAH, TRPM4 translocation and macroscopic current density increased significantly. Furthermore, TRPM4 accounted for a greater proportion of myogenic tone after SAH, suggesting an upregulation of TRPM4 activity in response to SAH. Cortical and global CBFs were reduced after SAH, but were restored significantly by 9-Phe, implying that TRPM4 contributed to CBF reduction after SAH. Collectively, these discoveries show that increased TRPM4 activity has a pivotal role in CBF reduction after SAH, and provide a novel target for the management of cerebral perfusion dysfunction following SAH.

TMEM16A is implicated in the regulation of coronary flow and is altered in hypertension

Coronary artery disease leads to ischaemic heart disease and ultimately myocardial infarction. Thus, it is important to determine the factors that regulate coronary blood flow. Ca2+ -activated chloride channels contribute to the regulation of arterial tone; however, their role in coronary arteries is unknown. The aim of this study was to investigate the expression and function of the main molecular correlate of Ca2+ -activated chloride channels, TMEM16A, in rat coronary arteries. We performed mRNA and protein analysis, electrophysiological studies of coronary artery myocytes, and functional studies of coronary artery contractility and coronary perfusion, using novel inhibitors of TMEM16A. Furthermore, we assessed whether any changes in expression and function occurred in coronary arteries from spontaneously hypertensive rats (SHRs). TMEM16A was expressed in rat coronary arteries. The TMEM16A-specific inhibitor, MONNA, hyperpolarised the membrane potential in U46619. MONNA, T16Ainh -A01, and Ani9 attenuated 5-HT/U46619-induced contractions. MONNA and T16Ainh -A01 also increased coronary flow in Langendorff perfused rat heart preparations. TMEM16A mRNA was increased in coronary artery smooth muscle cells from SHRs, and U46619 and 5-HT were more potent in arteries from SHRs than in those from normal Wistar rats. MONNA diminished this increased sensitivity to U46619 and 5-HT. In conclusion, TMEM16A is a key regulator of coronary blood flow and is implicated in the altered contractility of coronary arteries from SHRs.

Traumatic Brain Injury Alters BK Channel Subunit Expression, Localization, and Function in Cerebral Arterial Myocytes

Traumatic brain injury (TBI) induces acute changes in cerebrovascular function, which include alterations to cerebral artery myogenic tone and autoregulation. In arterial smooth muscle cells (myocytes), large‐conductance calcium‐activated potassium (BK) channels play a vital role in modulating plasma membrane potential and myogenic tone. Here, we examined BK channel expression and function in cerebral resistance size arteries one week following moderately‐severe TBI induced by controlled cortical impact with craniotomy. Total BKα (pore‐forming) and β1 (regulatory) subunit expression were reduced one week post‐TBI. Biotinylation of plasma membrane‐bound proteins revealed a compensatory increase of β1 subunits to the plasma membrane following TBI, which normalized single BK channel activation as assessed using patch‐clamp electrophysiology. However, the loss of total BKα subunits resulted in diminished whole cell, transient BK currents. Pressurized cerebral arteries from TBI rats exhibited greater myogenic tone at 60 mmHg and a diminished response to iberiotoxin, a BK channel pore blocker. In conclusion, moderately‐severe TBI enhances cerebral artery contractility, in part, through reductions in arterial myocyte BK channel subunit expression and function. Moreover, these TBI‐induced alterations in cerebral artery function can persist well beyond the initial 24–48 hour period post‐injury.Support or Funding InformationFlorida State UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Long Term Hypoxia Negatively Influences Ca2+ Signaling in Basilar Arterial Myocytes of Fetal and Adult Sheep

Gestational long term hypoxia (LTH) is a common prenatal stress caused by maternal anemia, high altitude living, smoking, and other disorders. Such gestational LTH can lead to a variety of cerebrovascular disorders in the neonate that compromise brain blood flow. Local and whole‐cell Ca2+ signals are important to the regulation of cerebrovascular tone. Rapid and localized Ca2+ transients, often referred to as sparks, activate large‐conductance K+ channels (BK), which dilate vessels. Whole‐cell Ca2+ oscillations, in comparison, are important to arterial contraction. Previous work shows that LTH and post‐natal maturity influence BK channel function in basilar arteries, arterial wall Ca2+ signals, cerebrovascular tone, as well as brain blood flow. We hypothesize that LTH‐dependent changes in spontaneous and depolarization mediated Ca2+ sparks and whole‐cell oscillations are important to BK channel activity, arterial wall Ca2+ signals, and vascular reactivity changes our group has previously observed. To begin evaluating the role of individual myocyte Ca2+ signals to the observed changes in cerebrovascular function, we isolated basilar arteries from adult and near term fetal (~141 gestational days) normoxic and hypoxic sheep that were raised at 3,800 m. for 110+ days. Cytosolic Ca2+ signals were examined in individual myocytes of intact arterial preparations loaded with fluo‐4 using laser scanning confocal microscopy techniques. Ca2+ spark activity as well as whole‐cell oscillations were enhanced by depolarizing myocytes with 30 mM K+. Maturation increased Ca2+ spark activity and depolarization‐dependent oscillations. LTH decreased oscillatory activity in fetal and adult animals but only spark frequency in adult myocytes. LTH decreased the area under the curve for Ca2+ oscillations independent of age as well as suppressed the ability of adult myocytes to respond to membrane depolarization. Depolarization, maturation, and LTH also influenced the spatial and temporal relationships of the Ca2+ oscillations. Overall, these observations illustrate that maturation and gestation at high‐altitude modify local and whole‐cell Ca2+ signaling, which likely contribute to adjustments in BK channel function, arterial wall Ca2+ signals, vasoreactivity, and cerebral blood flow that our group has examined in the past.Support or Funding InformationThis material is based upon work supported by NIH grants P01HD083132 (LZ). Imaging was performed in the LLUSM Advanced Imaging and Microscopy Core with support of NSF Grant MRI‐DBI 0923559 and the Loma Linda University School of Medicine.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chemokine (C‐X‐C motif) receptor 4 (CXCR4) activation in rat mesenteric artery myocytes is linked to suppression of Kv7 potassium channel activity

Chemokine (C‐X‐C motif) receptor 4 (CXCR4) expressed in arterial smooth muscle cells was recently implicated in the regulation of blood pressure. As a part of a heteromultimeric receptor complex with atypical chemokine receptor 3 and α1‐adrenergic receptors, CXCR4 modulates α1‐adrenergic signaling and function in vascular smooth muscle cells. Furthermore, activation of CXCR4 was found to sensitize rats to α1‐adrenergic receptor‐mediated blood pressure elevation. In neurons, α1‐adrenergic receptor activation has been linked to suppression of M‐currents mediated by Kv7 voltage‐gated potassium channels. Suppression of Kv7 currents has also been implicated as a mechanism of vasoconstrictor signal transduction downstream of Gq/11‐coupled receptor activation in arterial smooth muscle cells. We observed that the CXCR4 agonists CXCL12 (SDF‐1α, 10 nM and 100 nM) and ubiquitin (100 nM and 1 μM) significantly and dose‐dependently inhibit Kv7 potassium currents in freshly isolated mesenteric artery smooth muscle cells (MASMCs) by up to 50%. Pretreatment of MASMCs with the CXCR4 antagonist AMD3100 (1 μM) prevented inhibition of Kv7 current by CXCL12 and ubiquitin. Moreover, proximity ligation assays provided evidence for direct interactions of CXCR4 with Kv7.5 channel α‐subunits in MASMCs. Our findings suggest that Kv7 potassium channels are downstream effectors of CXCR4 receptor activation in arterial smooth muscle cells.Support or Funding InformationThe project described was supported by R01GM107495 (M. Majetschak, PI).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pregnancy Increases Ca2+ Sparks/Spontaneous Transient Outward Currents and Reduces Uterine Arterial Myogenic Tone.

Spontaneous transient outward currents (STOCs) at physiological membrane potentials of vascular smooth muscle cells fundamentally regulate vascular myogenic tone and blood flow in an organ. We hypothesize that heightened STOCs play a key role in uterine vascular adaptation to pregnancy. Uterine arteries were isolated from nonpregnant and near-term pregnant sheep. Ca2+ sparks were measured by confocal microscopy, and STOCs were determined by electrophysiological recording in smooth muscle cells. Percentage of Ca2+ spark firing myocytes increased dramatically at the resting condition in uterine arterial smooth muscle of pregnant animals, as compared with nonpregnant animals. Pregnancy upregulated the expression of RyRs (ryanodine receptors) and significantly boosted Ca2+ spark frequency. Ex vivo treatment of uterine arteries of nonpregnant sheep with estrogen and progesterone imitated pregnancy-induced RyR upregulation. STOCs occurred at much more negative membrane potentials in uterine arterial myocytes of pregnant animals. STOCs in uterine arterial myocytes were diminished by inhibiting large-conductance Ca2+-activated K+ (BKCa) channels and RyRs, thus functionally linking Ca2+ sparks and BKCa channel activity to STOCs. Pregnancy and steroid hormone treatment significantly increased STOCs frequency and amplitude in uterine arteries. Of importance, inhibition of STOCs with RyR inhibitor ryanodine eliminated pregnancy- and steroid hormone-induced attenuation of uterine arterial myogenic tone. Thus, the present study demonstrates a novel role of Ca2+ sparks and STOCs in the regulation of uterine vascular tone and provides new insights into the mechanisms underlying uterine vascular adaptation to pregnancy.

WP1-23 Vascular collagen 4A1 in subcortical white matter of older people and primates

ObjectivesTo test whether collagen 4A1 in cerebral small arteries associated with age, hypertension or small vessel disease (SVD).DesignNeuropathology cohort study.SubjectsOlder people age >65 years with minimal Alzheimer’s Disease.MethodsWe examined subcortical white matter in archived brain tissue from older people (n=34, 15F/19M, median age 84, range 65–99 y) and from experimental non-human primates (NHP, Macaca mulatta) that were young adults (n=9, age 6.2–8.3 y) or older adults (n=8, age 17.0–22.7 y). Some of the primates (5 young, 3 older) were chronically hypertensive. Vascular collagen 4A1 immunohistochemical labelling was examined qualitatively and quantified as percent area fraction.ResultsCollagen 4A1 labelling was common in arterial myocytes and in the adventitial layer in human and primate brain arteries, as well as in basement membrane, which frequently exhibited replication. Among older people, collagen 4A1 associated with neuropathological SVD severity (sclerotic index; r=−0.461, p=0.0409, least squares) and with radiological SVD severity (leukoaraiosis; p=0.0455, 1-way ANOVA) but not with age or clinical history of hypertension. In NHP, age but not hypertension was significantly associated with collagen-4A1 labelling (p=0.0396, 0.232 respectively, 2-way ANOVA).ConclusionsIn this small cohort, vascular collagen 4A1 was related to SVD severity in older humans, in accord with genetic associations of COL4A1 with SVD phenotypes.

Abstract WP154: Estrogen Modulates Serotonin-induced Vasoconstriction Through Tyrisine Kinase Inhibition

Earlier onset of menopause may influence the risk of stroke. However, hormone therapy, estrogen, alone or with a progestogen, increases stroke risk. Thus, it is needed to clarify whether estrogens prevent or promote vasomotor symptoms, as well as the mechanism(s) involved. Recent study showed that serotonin enhancing drugs, including antidepressants, migraine therapies and diet pills, can trigger a stroke via vasoconstriction. In this study, we examined the modulatory mechanisms of estrogen in serotonin -induced vasoconstriction by using a combinatory approach of isometric tension measurements, molecular biology, and patch-clamp techniques. 17β-estradiol (E2) elicited a significant and concentration-dependent relaxation of serotonin-induced contraction in deendothelialized aorta strips isolated from male rats. E2 triggered a relaxation of serotonin-induced contraction even in the presence of tamoxifen, an estrogen receptor antagonist, suggesting that E2-induced changes were not mediated by estrogen receptor. Patch clamp studies in rat arterial myocytes showed that E2 prevented Kv channel inhibition induced by serotonin. Serotonin increased Src activation in arterial smooth muscle required for contraction, which is significantly inhibited by E2. The estrogen receptor-independent inhibition of Src by E2 was confirmed in HEK239T cells that do not express estrogen receptor. Taken together, these results suggest that estrogen exerted vasodilatory effects on serotonin-precontracted artery via Src, implying a critical role of estrogen in prevention of vascular hyperreactivity to serotonin.

Preparation of Pulmonary Artery Myocytes and Rings to Study Vasoactive GPCRs.

G protein-coupled receptors (GPCR) are crucial transducers of extracellular signals into changes in vascular tone. Vasoactive GPCR stimulation in the pulmonary circuit may be elicited by agonists released in acute tissue hypoxia or inflammation, as well as chronic disease. Acute responses involve activation of smooth muscle contraction or relaxation machinery causing changes in actomyosin interaction, thereby altering lumen diameter. Chronic responses may typically include activation of proliferation or fibrosis. Using pulmonary artery myocytes and pulmonary artery rings, we describe a general strategy for quantification of vasoconstrictor or vasodilator GPCR responses, and for comparison of signaling pathways in cultured cells and in contracted vessels using immunohistochemistry of contracting vessels.

The cGMP-Degrading Enzyme Phosphodiesterase-5 (PDE5) in Cerebral Small Arteries of Older People.

Cerebral small vessel disease in deep penetrating arteries is a major cause of lacunar infarcts, white matter lesions and vascular cognitive impairment. Local cerebral blood flow in these small vessels is controlled by endothelial-derived nitric oxide, which exerts a primary vasodilator stimulus on vascular myocytes, via cytoplasmic cyclic GMP. Here, we investigated whether the cGMP-degrading enzyme phosphodiesterase-5 (PDE5) is present in small penetrating arteries in the deep subcortical white matter of older people. Frontal cortical tissue blocks were examined from donated brains of older people (n = 42, 24 male: 18 female, median age 81, range: 59-100 years). PDE5, detected by immunohistochemical labeling, was graded as absent, sparse, or abundant in vascular cells within small arteries in subcortical white matter (vessel outer diameter: 20-100 µm). PDE5 labeling within arterial myocytes was detected in all cases. Degree of PDE5 expression (absent, sparse, or abundant) was not associated with age or with neuropathological diagnosis of small vessel disease. In conclusion, PDE5 is present in vascular myocytes within small penetrating arteries in older people. This is a potential molecular target for pharmacological interventions.

Arterial smooth muscle cell PKD2 (TRPP1) channels regulate systemic blood pressure.

Systemic blood pressure is determined, in part, by arterial smooth muscle cells (myocytes). Several Transient Receptor Potential (TRP) channels are proposed to be expressed in arterial myocytes, but it is unclear if these proteins control physiological blood pressure and contribute to hypertension in vivo. We generated the first inducible, smooth muscle-specific knockout mice for a TRP channel, namely for PKD2 (TRPP1), to investigate arterial myocyte and blood pressure regulation by this protein. Using this model, we show that intravascular pressure and α1-adrenoceptors activate PKD2 channels in arterial myocytes of different systemic organs. PKD2 channel activation in arterial myocytes leads to an inward Na+ current, membrane depolarization and vasoconstriction. Inducible, smooth muscle cell-specific PKD2 knockout lowers both physiological blood pressure and hypertension and prevents pathological arterial remodeling during hypertension. Thus, arterial myocyte PKD2 controls systemic blood pressure and targeting this TRP channel reduces high blood pressure.

Estrogen modulates serotonin effects on vasoconstriction through Src inhibition

Estrogen has diverse effects on cardiovascular function, including regulation of the contractile response to vasoactive substances such as serotonin. The serotonin system recently emerged as an important player in the regulation of vascular tone in humans. However, hyperreactivity to serotonin appears to be a critical factor for the pathophysiology of hypertension. In this study, we examined the modulatory mechanisms of estrogen in serotonin-induced vasoconstriction by using a combinatory approach of isometric tension measurements, molecular biology, and patch-clamp techniques. 17β-Estradiol (E2) elicited a significant and concentration-dependent relaxation of serotonin-induced contraction in deendothelialized aortic strips isolated from male rats. E2 triggered a relaxation of serotonin-induced contraction even in the presence of tamoxifen, an estrogen receptor antagonist, suggesting that E2-induced changes are not mediated by estrogen receptor. Patch-clamp studies in rat arterial myocytes showed that E2 prevented Kv channel inhibition induced by serotonin. Serotonin increased Src activation in arterial smooth muscle required for contraction, which was significantly inhibited by E2. The estrogen receptor-independent inhibition of Src by E2 was confirmed in HEK293T cells that do not express estrogen receptor. Taken together, these results suggest that estrogen exerts vasodilatory effects on serotonin-precontracted arteries via Src, implying a critical role for estrogen in the prevention of vascular hyperreactivity to serotonin.

Na+/Ca2+ exchanger overexpression in smooth muscle augments cytosolic Ca2+ in femoral arteries of living mice.

Plasma membrane Na+/Ca2+ exchanger-1 (NCX1) helps regulate the cytosolic Ca2+ concentration ([Ca2+]CYT) in arterial myocytes. NCX1 mediates both Ca2+ entry and exit and tends to promote net Ca2+ entry in partially constricted arteries. Mean blood pressure (telemetry) is elevated by ≈10 mmHg in transgenic (TG) mice that overexpress NCX1 specifically in smooth muscle. We tested the hypothesis that NCX1 overexpression mediates Ca2+ gain and elevated [Ca2+]CYT in exposed femoral arteries that also express the Ca2+ biosensor exogenous myosin light chain kinase. [Ca2+]CYT and the NCX1-dependent (SEA0400-sensitive) component, ≈15% of total basal constriction in controls, were increased in TG arteries, but constrictions to phenylephrine and ANG II were comparable in TG and control arteries. Normalized phenylephrine dose-response curves and constriction to 30 and 300 ng/kg iv ANG II were virtually identical in control and TG arteries. ANG II-evoked constrictions, superimposed on elevated basal tone, accounted for the larger blood pressure responses to ANG II in TG arteries. TG and control mouse arteries fit the same pCa-constriction relationship over a wide range of pCa (≈125-500 nM). Vasodilation to acetylcholine, normalized to passive diameter, was also comparable in TG and control arteries, implying normal endothelial function. TG artery Na+ nitroprusside (nitric oxide donor)-induced dilations were, however, shifted to lower Na+ nitroprusside concentrations, indicating that TG myocyte vasodilator mechanisms were augmented. Maximum arterial dilation was comparable in TG and control mice, although passive diameter was ≈6-7% smaller in TG mice. The changes in TG arteries were apparently largely functional rather than structural, despite the congenital hypertension. NEW & NOTEWORTHY Smooth muscle Na+/Ca2+ exchanger-1 transgene overexpression (TG mice) increases femoral artery basal cytosolic Ca2+ concentration ([Ca2+]CYT) and tone in vivo and raises blood pressure. Arterial constriction to phenylephrine and angiotensin II are normal but superimposed on the augmented basal [Ca2+]CYT and tone (constriction) in TG mouse arteries. Similar effects in resistance arteries would explain the elevated blood pressure. Acetylcholine-induced vasodilation is unimpaired, implying a normal endothelium, but TG arteries are hypersensitive to sodium nitroprusside.

Melatonin activates BKCa channels in cerebral artery myocytes via both direct and MT receptor/PKC-mediated pathway

The pineal hormone melatonin is a neuroendocrine hormone with high membrane permeability that is involved in regulation of circadian rhythm of several biological functions. Large-conductance Ca2+-activated K+ (BKCa) channels are abundantly expressed in vascular smooth muscle cells and play an important role in vascular tone regulation. We investigated the mechanisms through which myocyte BKCa channels mediate effects of melatonin on cerebral arteries (CAs). Arterial contractility measurements showed that melatonin alone did not change vascular tone in CAs; however, it induced concentration-dependent vasodilation of phenylephrine-induced contraction in CAs. In the presence of the potent endothelial oxide synthase inhibitor, Nω-nitro-L-arginine methyl ester, melatonin-elicited relaxation was significantly inhibited by iberiotoxin (BKCa channel blocker). Melatonin significantly increased BKCa currents but not voltage-gated K+ (KV) currents in whole-cell recordings. Melatonin decreased the amplitude of Ca2+ sparks and spontaneous transient outward currents (STOCs), however, a significant increase in open probability of BKCa channels was observed in both inside-out and cell-attached patch-clamp recordings. This melatonin-induced enhancement of BKCa channel activity was significantly suppressed by luzindole (melatonin MT1/MT2 receptor inhibitor), U73122 (phospholipase C (PLC) inhibitor), and Ro31–8220 (protein kinase C (PKC) inhibitor). Melatonin had no significant effects on sarcoplasmic reticulum release of Ca2+. These findings indicate that melatonin-induced vasorelaxation of CAs is partially attributable to direct (passing through the cell membrane) and indirect (via melatonin MT1/MT2 receptors-PLC-PKC pathway) activation of BKCa channels on CA myocytes.

PO-137 Epigenetic regulation of exercise-improved LTCC and BKCa channels function in hypertension mesenteric arteries

Objective To investigate the epigenetic mechanism of the voltage-gated L-type Ca2+ channel (LTCC) and the large-conductance Ca2+-activated K+ channel (BKCa) function in mesenteric arterial myocytes improved by regular aerobic exercise in hypertension.
 Methods 12-week-old male SHR and WKY rats were randomly assigned to sedentary and exercise training groups, respectively. Exercise groups were performed a moderate-intensity treadmill running. After 8 weeks, patch clamp study, Ca2+ image, Western blot, qPCR, bisulfite sequencing PCR were used to detect the LTCC and BKCa channel currents, BKCa single channel gating properties, Ca2+ spark, mRNA and protein expression of LTCC α1c together with BKCa α and β1 subunits, DNA methylation level of α1c and β1 gene promoter region, miR-328 expression. In vitro experiment，miR-328 mimic and miR-328 inhibitor were transfected into cultured arterial myocytes to make miR-328 overexpressing or silencing, the mRNA and protein expression of α1c subunits were determined after 48 h transfection.
 Results 1) After 8 weeks of exercise, SBP in both exercise groups of WKY and SHR were significantly lower than that of their sedentary counterparts. 2) Exercise normalized the increased LTCC and BKCa current density of mesenteric arterial myocytes in SHR. 3) Exercise attenuated the increased single BKCa channel open Probability (Po) and the amplitude of Ca2+ spark in hypertension. 4) Exercise inhibited the upregulated mRNA and protein expression of BKCa β1 subunit in mesenteric arteries from SHR; β1 gene promoter was demethylation in hypertension, exercise increased the methylation level at β1 gene promoter of SHR. 5) The protein expression of LTCC α1c subunit was significantly increased in SHR, while decreased by exercise; the expression of miR-328 in mesenteric arteries was highly negative correlation with α1c subunit. 6) The miR-328 overexpression by transfecting miR-328 mimic decreased α1c subunit protein level significantly, while miR-328 inhibitor made α1c subunit a slight increase.
 Conclusions Regular aerobic exercise efficiently reduces blood pressure of SHR, enhances β1 gene promoter methylation, mediates miR-328 inhibiting the α1c expression at post-transcriptional level, which might be the epigenetic mechanism underlying exercise-improved LTCC and BKCa channels function in mesenteric arteries of hypertension.

The LKB1-AMPK-α1 signaling pathway triggers hypoxic pulmonary vasoconstriction downstream of mitochondria.

Hypoxic pulmonary vasoconstriction (HPV), which aids ventilation-perfusion matching in the lungs, is triggered by mechanisms intrinsic to pulmonary arterial smooth muscles. The unique sensitivity of these muscles to hypoxia is conferred by mitochondrial cytochrome c oxidase subunit 4 isoform 2, the inhibition of which has been proposed to trigger HPV through increased generation of mitochondrial reactive oxygen species. Contrary to this model, we have shown that the LKB1-AMPK-α1 signaling pathway is critical to HPV. Spectral Doppler ultrasound revealed that deletion of the AMPK-α1 catalytic subunit blocked HPV in mice during mild (8% O2) and severe (5% O2) hypoxia, whereas AMPK-α2 deletion attenuated HPV only during severe hypoxia. By contrast, neither of these genetic manipulations affected serotonin-induced reductions in pulmonary vascular flow. HPV was also attenuated by reduced expression of LKB1, a kinase that activates AMPK during energy stress, but not after deletion of CaMKK2, a kinase that activates AMPK in response to increases in cytoplasmic Ca2+ Fluorescence imaging of acutely isolated pulmonary arterial myocytes revealed that AMPK-α1 or AMPK-α2 deletion did not affect mitochondrial membrane potential during normoxia or hypoxia. However, deletion of AMPK-α1, but not of AMPK-α2, blocked hypoxia from inhibiting KV1.5, the classical "oxygen-sensing" K+ channel in pulmonary arterial myocytes. We conclude that LKB1-AMPK-α1 signaling pathways downstream of mitochondria are critical for the induction of HPV, in a manner also supported by AMPK-α2 during severe hypoxia.