Arterial smooth muscle Na
/Ca
exchanger-1 (SM-NCX1) promotes vasoconstriction or vasodilation by mediating, respectively, Ca
influx or efflux. In vivo, SM-NCX1 mediates net Ca
influx to help maintain ...myogenic tone (MT) and neuronally activated constriction. SM-NCX1-TG (overexpressing transgenic) mice have increased MT and mean blood pressure (MBP; +13.5 mmHg); SM-NCX1-KO (knockout) mice have reduced MT and MBP (-11.1 mmHg). Endothelium-dependent vasodilation (EDV) is often impaired in hypertension. We tested whether genetically engineered SM-NCX1 expression and consequent BP changes similarly alter EDV. Isolated, pressurized mesenteric resistance arteries with MT from SM-NCX1-TG and conditional SM-NCX1-KO mice, and femoral arteries in vivo from TG mice were studied. Acetylcholine (ACh)-dilated TG arteries with MT slightly more than control or KO arteries, implying that SM-NCX1 overexpression does not impair EDV. In preconstricted KO, but not TG mouse arteries, however, ACh- and bradykinin-triggered vasodilation was markedly attenuated. To circumvent the endothelium, phenylephrine-constricted resistance arteries were tested with Na-nitroprusside SNP; nitric oxide (NO) donor and cGMP. This endothelium-independent vasodilation was augmented in TG but attenuated in KO arteries that lack NCX1-mediated Ca
clearance. Baseline cytosolic Ca
(Ca
) was elevated in TG femoral arteries in vivo, supporting the high BP; furthermore, SNP-triggered Ca
decline and vasodilation were augmented as NO and cGMP promote myocyte polarization thereby enhancing NCX1-mediated Ca
efflux. The TG mouse data indicate that BP elevation does not attenuate endothelium-dependent vasodilation. Thus, in essential hypertension and many models the endothelial impairment that supports the hypertension apparently is not triggered by BP elevation but by extravascular mechanisms.
Endothelium-dependent, ACh-induced vasodilation (EDV) is attenuated, and arterial myocyte Na
/Ca
exchangers (NCX1) are upregulated in many forms of hypertension. Surprisingly, mildly hypertensive smooth muscle-specific (SM)-NCX1 transgenic mice exhibited modestly enhanced EDV and augmented endothelium-independent vasodilation (EIV). Conversely, mildly hypotensive SM-NCX1-knockout mice had greatly attenuated EIV. These adaptations help compensate for NCX1 expression-induced alterations in cytosolic Ca
and blood pressure (BP) and belie the view that elevated BP, itself, causes the endothelial dysregulation in hypertension.
Excess dietary salt is a major cause of hypertension. Nevertheless, the specific mechanisms by which salt increases arterial constriction and peripheral vascular resistance, and thereby raises blood ...pressure (BP), are poorly understood. Here we summarize recent evidence that defines specific molecular links between Na(+) and the elevated vascular resistance that directly produces high BP. In this new paradigm, high dietary salt raises cerebrospinal fluid Na(+). This leads, via the Na(+)-sensing circumventricular organs of the brain, to increased sympathetic nerve activity (SNA), a major trigger of vasoconstriction. Plasma levels of endogenous ouabain (EO), the Na(+) pump ligand, also become elevated. Remarkably, high cerebrospinal fluid Na(+)-evoked, locally secreted (hypothalamic) EO participates in a pathway that mediates the sustained increase in SNA. This hypothalamic signaling chain includes aldosterone, epithelial Na(+) channels, EO, ouabain-sensitive α(2) Na(+) pumps, and angiotensin II (ANG II). The EO increases (e.g.) hypothalamic ANG-II type-1 receptor and NADPH oxidase and decreases neuronal nitric oxide synthase protein expression. The aldosterone-epithelial Na(+) channel-EO-α(2) Na(+) pump-ANG-II pathway modulates the activity of brain cardiovascular control centers that regulate the BP set point and induce sustained changes in SNA. In the periphery, the EO secreted by the adrenal cortex directly enhances vasoconstriction via an EO-α(2) Na(+) pump-Na(+)/Ca(2+) exchanger-Ca(2+) signaling pathway. Circulating EO also activates an EO-α(2) Na(+) pump-Src kinase signaling cascade. This increases the expression of the Na(+)/Ca(2+) exchanger-transient receptor potential cation channel Ca(2+) signaling pathway in arterial smooth muscle but decreases the expression of endothelial vasodilator mechanisms. Additionally, EO is a growth factor and may directly participate in the arterial structural remodeling and lumen narrowing that is frequently observed in established hypertension. These several central and peripheral mechanisms are coordinated, in part by EO, to effect and maintain the salt-induced elevation of BP.
Myocardial ischemia/reperfusion (I/R) induces inflammatory response that may lead to remote vascular injury. Vagal nerve elicits the cholinergic anti-inflammatory pathway by activating α7 nicotinic ...acetylcholine receptors (α7nAChR). Nevertheless, the role of vagal nerve-mediated anti-inflammatory pathway in the vasculature has not been studied previously. Therefore, we aimed to clarify the potential role of vagal stimulation (VNS) in regulating remote vascular injury after myocardial I/R. Adult male Sprague–Dawley rats were subjected to VNS starting 15 min prior to ischemia until the end of reperfusion. VNS not only reduced infarct size and improved cardiac function, but also ameliorated myocardial I/R-induced dysfunctional vasoconstriction and vasodilatation and degradation of endothelial structure in mesenteric arteries. VNS decreased serum and vascular levels of tumor necrosis factor-α and IL-1β. Interestingly, in vivo microdialysis studies demonstrated that VNS increased ACh concentration in the mesenteric circulation. Furthermore, VNS up-regulated expressions of muscarinic ACh receptors-3 (M
3
AChR) and α7nAChR in mesenteric arteries. Preserved endothelial relaxations by VNS were inhibited by atropine or methyllycaconitine, indicating that functional protection was associated with M
3
and α7nAChR activation. Finally, VNS increased STAT3 phosphorylation and inhibited NF-κB activation in mesenteric arteries, and these effects were abolished by α7nAChR shRNA treatment, indicating VNS-mediated anti-inflammatory effect mainly involved α7nAChR. These results demonstrated for the first time that VNS protected against remote vascular dysfunction, through the cholinergic anti-inflammatory pathway which is dependent on α7nAChR. Our findings represent a significant addition to the understanding of vagal nerve-mediated pathways and the potential roles they play in regulating the vasculature.
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.
Ca
signaling, particularly the mechanism via store-operated Ca
entry (SOCE) and receptor-operated Ca
entry (ROCE), plays a critical role in the development of acute hypoxia-induced pulmonary ...vasoconstriction and chronic hypoxia-induced pulmonary hypertension. This study aimed to test the hypothesis that chronic hypoxia differentially regulates the expression of proteins that mediate SOCE and ROCE stromal interacting molecule (STIM), Orai, and canonical transient receptor potential channel TRPC6 in pulmonary (PASMC) and coronary (CASMC) artery smooth muscle cells. The resting cytosolic Ca
(Ca
) and the stored Ca
in the sarcoplasmic reticulum were not different in CASMC and PASMC. Seahorse measurement showed a similar level of mitochondrial bioenergetics (basal respiration and ATP production) between CASMC and PASMC. Glycolysis was significantly higher in PASMC than in CASMC. The amplitudes of cyclopiazonic acid-induced SOCE and OAG-induced ROCE in CASMC are slightly, but significantly, greater than in PASMC. The frequency and the area under the curve of Ca
oscillations induced by ATP and histamine were also larger in CASMC than in PASMC. Na
/Ca
exchanger-mediated increases in Ca
did not differ significantly between CASMC and PASMC. The basal protein expression levels of STIM1/2, Orai1/2, and TRPC6 were higher in CASMC than in PASMC, but hypoxia (3% O
for 72 h) significantly upregulated protein expression levels of STIM1/STIM2, Orai1/Orai2, and TRPC6 and increased the resting Ca
only in PASMC, but not in CASMC. The different response of essential components of store-operated and receptor-operated Ca
channels to hypoxia is a unique intrinsic property of PASMC, which is likely one of the important explanations why hypoxia causes pulmonary vasoconstriction and induces pulmonary vascular remodeling, but causes coronary vasodilation.
Reduced smooth muscle (SM)‐specific α2 Na+ pump expression elevates basal blood pressure (BP) and increases BP sensitivity to angiotensin II (Ang II) and dietary NaCl, whilst SM‐α2 overexpression ...lowers basal BP and decreases Ang II/salt sensitivity. Prolonged ouabain infusion induces hypertension in rodents, and ouabain‐resistant mutation of the α2 ouabain binding site (α2R/R mice) confers resistance to several forms of hypertension. Pressure overload‐induced heart hypertrophy and failure are attenuated in cardio‐specific α2 knockout, cardio‐specific α2 overexpression and α2R/R mice. We propose a unifying hypothesis that reconciles these apparently disparate findings: brain mechanisms, activated by Ang II and high NaCl, regulate sympathetic drive and a novel neurohumoral pathway mediated by both brain and circulating endogenous ouabain (EO). Circulating EO modulates ouabain‐sensitive α2 Na+ pump activity and Ca2+ transporter expression and, via Na+/Ca2+ exchange, Ca2+ homeostasis. This regulates sensitivity to sympathetic activity, Ca2+ signalling and arterial and cardiac contraction.
The centrally controlled, parallel sympathetic nerve and slow neurohumoral pathways that regulate both arterial and cardiac function and participate in the pathogenesis of hypertension and heart failure (HF). Angiotensin II (Ang II) and high dietary salt are convergent signals that act via hypothalamic Ang type 1 receptors (AT1R) to activate CNS sympathetic pathways. The increased sympathetic nerve activity (SNA) promotes vasoconstriction and increases cardiac rate and contractile force. Prolonged stimulation of hypothalamic AT1Rs also activates a novel neurohumoral pathway (box at upper right) that includes aldosterone (Aldo), mineralocorticoid receptors (MR), epithelial Na+ channels (ENaC), endogenous ouabain (EO) and α2 Na+ pumps. This hypothalamic pathway feeds back (dashed green line, ‘+’) to modulate Ang II‐activated SNA and also promotes adrenal secretion of EO, triggered by, e.g., ACTH, adrenal SNA and/or Ang II. The elevated plasma EO acutely inhibits α2 Na+ pumps (NKAs) in both the heart and arteries, and the rise in intracellular Na+ rapidly induces Na+/Ca2+ exchanger (NCX)‐mediated Ca2+ gain, and cardiotonic and vasotonic effects. Prolonged plasma EO elevation also activates an α2 Na+ pump‐associated protein kinase cascade (e.g. C‐Src‐mediated) that increases cardiomyocyte (CMC) and arterial smooth muscle cell (ASMC) NCX expression, and arterial sarcoplasmic reticulum (SR) Ca2+ pump (SERCA2) expression. In arteries with tone, NCX normally favours Ca2+ entry and helps to sustain cytosolic Ca2+ (Ca2+CYT) above contraction threshold. The EO‐induced NCX and SERCA2 up‐regulation enhance Ca2+ signalling and help the very modestly increased SNA to increase vascular tone and resistance, and elevate blood pressure. In the heart, NCX promotes Ca2+ extrusion during diastole, but prolonged α2 pump inhibition by EO reduces the Na+ gradient driving force so that Na+CYT and diastolic Ca2+CYT are both elevated; consequently, cardiac relaxation is slow and/or incomplete. Also, cardiac SERCA2 expression is usually reduced in HF (perhaps due to the high EO), as are SR Ca2+ stores and Ca2+ transients, and systolic function is impaired. The diastolic dysfunction and attenuated cardiac contraction and stroke volume help explain HF. This review describes research on mice with genetically engineered α2 Na+ pumps and related studies that elucidate these cellular mechanisms.
Plasma membrane Na
/Ca
exchanger-1 (NCX1) helps regulate the cytosolic Ca
concentration (Ca
) in arterial myocytes. NCX1 mediates both Ca
entry and exit and tends to promote net Ca
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 Ca
gain and elevated Ca
in exposed femoral arteries that also express the Ca
biosensor exogenous myosin light chain kinase. Ca
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
/Ca
exchanger-1 transgene overexpression (TG mice) increases femoral artery basal cytosolic Ca
concentration (Ca
) and tone in vivo and raises blood pressure. Arterial constriction to phenylephrine and angiotensin II are normal but superimposed on the augmented basal Ca
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.
OBJECTIVE—We explored the role of endoplasmic reticulum (ER)–mitochondria Ca cross talk involving voltage-dependent anion channel-1 (VDAC1)/glucose-regulated protein 75/inositol 1,4,5-trisphosphate ...receptor 1 complex and mitofusin 2 in endothelial cells during hypoxia/reoxygenation (H/R), and investigated the protective effects of acetylcholine.
APPROACH AND RESULTS—Acetylcholine treatment during reoxygenation prevented intracellular and mitochondrial Ca increases and alleviated ER Ca depletion during H/R in human umbilical vein endothelial cells. Consequently, acetylcholine enhanced mitochondrial membrane potential and inhibited proapoptotic cascades, thereby reducing cell death and preserving endothelial ultrastructure. This effect was likely mediated by the type-3 muscarinic acetylcholine receptor and the phosphatidylinositol 3-kinase/Akt pathway. In addition, interactions among members of the VDAC1/glucose-regulated protein 75/inositol 1,4,5-trisphosphate receptor 1 complex were increased after H/R and were associated with mitochondrial Ca overload and cell death. Inhibition of the partner of the Ca channeling complex (VDAC1 siRNA) or a reduction in ER–mitochondria tethering (mitofusin 2 siRNA) prevented the increased protein interaction within the complex and reduced mitochondrial Ca accumulation and subsequent endothelial cell death after H/R. Intriguingly, acetylcholine could modulate ER–mitochondria Ca cross talk by inhibiting the VDAC1/glucose-regulated protein 75/inositol 1,4,5-trisphosphate receptor 1 complex and mitofusin 2 expression. Phosphatidylinositol 3-kinase siRNA diminished acetylcholine-mediated inhibition of mitochondrial Ca overload and VDAC1/glucose-regulated protein 75/inositol 1,4,5-trisphosphate receptor 1 complex formation induced by H/R.
CONCLUSIONS—Our data suggest that ER–mitochondria interplay plays an important role in reperfusion injury in the endothelium and may be a novel molecular target for endothelial protection. Acetylcholine attenuates both intracellular and mitochondrial Ca overload and protects endothelial cells from H/R injury, presumably by disrupting the ER–mitochondria interaction.
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