NO-induced vasodilation correlates directly with BP in smooth muscle-Na/Ca exchanger-1-engineered mice: elevated BP does not attenuate endothelial function.

Arterial smooth muscle Na+/Ca2+ exchanger-1 (SM-NCX1) promotes vasoconstriction or vasodilation by mediating, respectively, Ca2+ influx or efflux. In vivo, SM-NCX1 mediates net Ca2+ 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 Ca2+ clearance. Baseline cytosolic Ca2+ ([Ca2+]cyt) was elevated in TG femoral arteries in vivo, supporting the high BP; furthermore, SNP-triggered [Ca2+]cyt decline and vasodilation were augmented as NO and cGMP promote myocyte polarization thereby enhancing NCX1-mediated Ca2+ 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.NEW & NOTEWORTHY Endothelium-dependent, ACh-induced vasodilation (EDV) is attenuated, and arterial myocyte Na+/Ca2+ 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 Ca2+ and blood pressure (BP) and belie the view that elevated BP, itself, causes the endothelial dysregulation in hypertension.

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.

Hypoxia selectively upregulates cation channels and increases cytosolic [Ca2+] in pulmonary, but not coronary, arterial smooth muscle cells.

Ca2+ signaling, particularly the mechanism via store-operated Ca2+ entry (SOCE) and receptor-operated Ca2+ 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 [Ca2+] ([Ca2+]cyt) and the stored [Ca2+] 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 Ca2+ oscillations induced by ATP and histamine were also larger in CASMC than in PASMC. Na+/Ca2+ exchanger-mediated increases in [Ca2+]cyt 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% O2 for 72 h) significantly upregulated protein expression levels of STIM1/STIM2, Orai1/Orai2, and TRPC6 and increased the resting [Ca2+]cyt only in PASMC, but not in CASMC. The different response of essential components of store-operated and receptor-operated Ca2+ 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.

Pivotal role of α2 Na+ pumps and their high affinity ouabain binding site in cardiovascular health and disease.

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.

Reduction of Mitochondria-Endoplasmic Reticulum Interactions by Acetylcholine Protects Human Umbilical Vein Endothelial Cells From Hypoxia/Reoxygenation Injury.

OBJECTIVE: We explored the role of endoplasmic reticulum (ER)-mitochondria Ca(2+) 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(2+) increases and alleviated ER Ca(2+) 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(2+) overload and cell death. Inhibition of the partner of the Ca(2+) 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(2+) accumulation and subsequent endothelial cell death after H/R. Intriguingly, acetylcholine could modulate ER-mitochondria Ca(2+) 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(2+) 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(2+) overload and protects endothelial cells from H/R injury, presumably by disrupting the ER-mitochondria interaction.

Intravital Förster resonance energy transfer imaging reveals elevated [Ca2+]i and enhanced sympathetic tone in femoral arteries of angiotensin II-infused hypertensive biosensor mice.

Artery narrowing in hypertension can only result from structural remodelling of the artery, or increased smooth muscle contraction. The latter may occur with, or without, increases in [Ca(2+)]i. Here, we sought to measure, in living hypertensive mice, possible changes in artery dimensions and/or [Ca(2+)]i, and to determine some of the mechanisms involved. Ca(2+)/calmodulin biosensor (Förster resonance energy transfer-based) mice were made hypertensive by s.c. infusion of angiotensin II (Ang II, 400 ng kg(-1) min(-1), 2-3 weeks). Intravital fluorescence microscopy was used to determine [Ca(2+)]i and outer diameter of surgically exposed, intact femoral artery (FA) of anaesthetized mice. Active contractile FA 'tone' was calculated from the basal-state diameter and the passive (i.e. Ca(2+)-free) diameter (PD). Compared to saline control, FAs of Ang II-infused mice had (1) ∼21% higher active tone and (2) ∼78 nm higher smooth muscle [Ca(2+)]i, but (3) the same PDs. The local Ang II receptor (AT1R) blocker losartan had negligible effect on tone or [Ca(2+)]i in control FAs, but reduced the basal tone by ∼9% in Ang II FAs. Both i.v. hexamethonium and locally applied prazosin abolished the difference in FA tone and [Ca(2+)]i, suggesting a dominant role of sympathetic nerve activity (SNA). Changes in diameter and [Ca(2+)]i in response to locally applied phenylephrine, Ang II, arginine vasopressin, elevated [K(+)]o and acetylcholine were not altered. In summary, FAs of living Ang II hypertensive mice have higher [Ca(2+)]i, and are more constricted, due, primarily, to elevated SNA and some increased arterial AT1R activation. Evidence of altered artery reactivity or remodeling was not found.

How NaCl raises blood pressure: a new paradigm for the pathogenesis of salt-dependent 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.

Endothelial dysfunction in rat mesenteric artery after regional cardiac ischaemia-reperfusion.

In most previous studies, ischaemia-reperfusion (I/R)-induced vascular injury referred to injury in the tissue or blood vessel that was directly subjected to I/R. However, less attention has been focused on remote vascular injury that might be caused by cardiac I/R. In the present study, we aimed to assess whether cardiac I/R could affect vasoconstriction and vasodilatation in mesenteric arteries from Sprague-Dawley rats. Left anterior descending coronary arteries from adult male Sprague-Dawley rats were occluded (60 min) and then reperfused (120 min). Changes in haemodynamic parameters indicated that this procedure caused evident cardiac dysfunction. In mesenteric arteries isolated from the animals, cardiac I/R significantly increased the maximal contractions in response to KCl, 5-hydroxytryptamine, phenylephrine and U46619 and decreased the maximal relaxation in response to acetylcholine, but not to sodium nitroprusside, compared with sham-operated animals. The nitric oxide synthase inhibitor L-NAME abolished differences of contractile responses to phenylephrine between sham-operated and I/R rats. The antioxidant N-acetyl-L-cysteine reversed the impairment of acetylcholine-stimulated vasodilatation induced by regional cardiac I/R. However, L-NAME caused a similar degree of inhibition of acetylcholine-stimulated relaxation in mesenteric arteries from sham-operated and I/R rats. Electron microscopy revealed that mesenteric arterial endothelial structure was degraded in the I/R group and that N-acetyl-L-cysteine treatment prevented this structural damage. In conclusion, regional cardiac I/R caused by transient occlusion and reperfusion of the left anterior descending coronary artery results in peripheral vascular endothelial dysfunction.

Tumor necrosis factor-α enhances microvascular tone and reduces blood flow in the cochlea via enhanced sphingosine-1-phosphate signaling.

BACKGROUND AND PURPOSE: We sought to demonstrate that tumor necrosis factor (TNF)-α, via sphingosine-1-phosphate signaling, has the potential to alter cochlear blood flow and thus, cause ischemic hearing loss. METHODS: We performed intravital fluorescence microscopy to measure blood flow and capillary diameter in anesthetized guinea pigs. To measure capillary diameter ex vivo, capillary beds from the gerbil spiral ligament were isolated from the cochlear lateral wall and maintained in an organ bath. Isolated gerbil spiral modiolar arteries, maintained and transfected in organ culture, were used to measure calcium sensitivity (calcium-tone relationship). In a clinical study, a total of 12 adult patients presenting with typical symptoms of sudden hearing loss who were not responsive or only partially responsive to prednisolone treatment were identified and selected for etanercept treatment. Etanercept (25 mg s.c.) was self-administered twice a week for 12 weeks. RESULTS: TNF-α induced a proconstrictive state throughout the cochlear microvasculature, which reduced capillary diameter and cochlear blood flow in vivo. In vitro isolated preparations of the spiral modiolar artery and spiral ligament capillaries confirmed these observations. Antagonizing sphingosine-1-phosphate receptor 2 subtype signaling (by 1 µmol/L JTE013) attenuated the effects of TNF-α in all models. TNF-α activated sphingosine kinase 1 (Sk1) and induced its translocation to the smooth muscle cell membrane. Expression of a dominant-negative Sk1 mutant (Sk1(G82D)) eliminated both baseline spiral modiolar artery calcium sensitivity and TNF-α effects, whereas a nonphosphorylatable Sk1 mutant (Sk1(S225A)) blocked the effects of TNF-α only. A small group of etanercept-treated, hearing loss patients recovered according to a 1-phase exponential decay (half-life=1.56 ± 0.20 weeks), which matched the kinetics predicted for a vascular origin. CONCLUSIONS: TNF-α indeed reduces cochlear blood flow via activation of vascular sphingosine-1-phosphate signaling. This integrates hearing loss into the family of ischemic microvascular pathologies, with implications for risk stratification, diagnosis, and treatment.

Sympathetic nerves and the endothelium influence the vasoconstrictor effect of low concentrations of ouabain in pressurized small arteries.

We hypothesized that in salt-dependent forms of hypertension, endogenous ouabain acts on arterial smooth muscle to cause enhanced vasoconstriction. Here, we tested for the involvement of the arterial endothelium and perivascular sympathetic nerve terminals in ouabain-induced vasoconstriction. Segments of rat mesenteric or renal interlobar arteries were pressurized to 70 mmHg at 37 degrees C and exposed to ouabain (10(-11)-10(-7) M). Removal of the endothelium enhanced ouabain-induced vasoconstriction by as much as twofold (at an ouabain concentration of 10(-9) M). A component of the ouabain-induced vasoconstriction is due to the enhanced spontaneous release of norepinephrine (NE) from nerve terminals in the arterial wall. The alpha(1)-adrenoceptor blocker prazosin (10(-6) M) decreased ouabain-induced vasoconstrictions by as much as 50%. However, neither the contraction induced by sympathetic nerve activity (SNA) nor the NE release evoked by SNA (measured directly by carbon fiber amperometry) was increased by ouabain (<10(-7) M). Nevertheless, the converse case was true: after brief bursts of SNA, vasoconstrictor responses to ouabain were transiently increased (1.75-fold). This effect may be mediated by neuropeptide Y and Y(1) receptors on smooth muscle. In arteries lacking the endothelium and exposed to prazosin, ouabain (10(-11) M and greater) caused vasoconstriction, indicating a direct effect of very "low" concentrations of ouabain on arterial smooth muscle. In conclusion, in intact arteries, the endothelium opposes ouabain (10(-11)-10(-7)M)-induced vasoconstriction, which is caused by both enhanced spontaneous NE release and direct effects on smooth muscle. Ouabain (<10(-7)M) does not enhance SNA-mediated contractions, but SNA enhances ouabain-induced contractions. The effects of endogenous ouabain may be accentuated in forms of hypertension that involve sympathetic nerve hyperactivity and/or endothelial dysfunction.

In vivo assessment of artery smooth muscle [Ca2+]i and MLCK activation in FRET-based biosensor mice.

The cellular mechanisms that control arterial diameter in vivo, particularly in hypertension, are uncertain. Here, we report a method that permits arterial intracellular Ca(2+) concentration ([Ca(2+)](i)), myosin light-chain kinase (MLCK) activation, and artery external diameter to be recorded simultaneously with arterial blood pressure (BP) in living mice under 1.5% isofluorane anesthesia. The method also enables an assessment of local receptor activity on [Ca(2+)](i), MLCK activity, and diameter in arteries, uncomplicated by systemic effects. Transgenic mice that express, in smooth muscle, a Ca(2+)/calmodulin-activated, Förster resonance energy transfer (FRET)-based "ratiometric", exogenous MLCK biosensor were used. Vasoactive substances were administered either intravenously or locally to segments of exposed femoral or cremaster arteries. In the basal state, mean BP was approximately 90 mmHg, femoral arteries were constricted to 65% of their passive diameter, MLCK fractional activation was 0.14, and [Ca(2+)](i) was 131 nM. Phenylephrine (300 ng/g wt iv) elevated mean BP transiently to approximately 110 mmHg, decreased heart rate, increased femoral artery [Ca(2+)](i) to 244 nM and fractional MLCK activation to 0.24, and decreased artery diameter by 23%. In comparison, local application of 1.0 muM phenylephrine raised [Ca(2+)](i) to 279 nM and fractional MLCK activation to 0.26, and reduced diameter by 25%, but did not affect BP or heart rate. Intravital FRET imaging of exogenous MLCK biosensor mice permits quantification of changes in [Ca(2+)](i) and MLCK activation that accompany small changes in BP. Based on the observed variance of the FRET data, this method should enable the detection of a difference in basal [Ca(2+)](i) of 29 nM between two groups of 12 mice with a significance of P < 0.05.

Exercise improves the dilatation function of mesenteric arteries in postmyocardial infarction rats via a PI3K/Akt/eNOS pathway-mediated mechanism.

Myocardial infarction (MI) has been shown to induce endothelial dysfunction in peripheral resistance arteries and thus increase peripheral resistance. This study was designed to investigate the underlying mechanisms of post-MI-related dysfunctional dilatation of peripheral resistance arteries and, furthermore, to examine whether exercise may restore dysfunctional dilatation of peripheral resistance arteries. Adult male Sprague-Dawley rats were divided into three groups: sham-operated, MI, and MI + exercise. Ultrastructure and relaxation function of the mesenteric arteries, as well as phosphatidylinositol-3 kinase (PI3K), Akt kinases (Akt), endothelial nitric oxide synthase (eNOS) activity, and phosphorylation of PI3K, Akt, and eNOS by ACh were determined. Post-MI rats exhibited pronounced ultrastructural changes in mesenteric artery endothelial cells and endothelial dysfunction. In addition, the activities of PI3K, Akt, and eNOS, and their phosphorylation by ACh were significantly attenuated in mesenteric arteries (P < 0.05-0.01). After 8 wk of exercise, not only did endothelial cells appeared more normal in structure, but also ameliorated post-MI-associated mesenteric arterial dysfunction, which were accompanied by elevated activities of PI3K, Akt, and eNOS, and their phosphorylation by ACh (P < 0.05-0.01). Importantly, inhibition of either PI3K or eNOS attenuated exercise-induced restoration of the dilatation function and blocked PI3K, Akt, and eNOS phosphorylation by ACh in the mesenteric arteries. These data demonstrate that MI induces dysfunctional dilation of peripheral resistance arteries by degradation of endothelial structural integrity and attenuating PI3K-Akt-eNOS signaling. Exercise may restore dilatation function of peripheral resistance arteries by protecting endothelial structural integrity and increasing PI3K-Akt-eNOS signaling cascades.

Knockout of Na+/Ca2+ exchanger in smooth muscle attenuates vasoconstriction and L-type Ca2+ channel current and lowers blood pressure.

Mice with smooth muscle (SM)-specific knockout of Na(+)/Ca(2+) exchanger type-1 (NCX1(SM-/-)) and the NCX inhibitor, SEA0400, were used to study the physiological role of NCX1 in mouse mesenteric arteries. NCX1 protein expression was greatly reduced in arteries from NCX1(SM-/-) mice generated with Cre recombinase. Mean blood pressure (BP) was 6-10 mmHg lower in NCX1(SM-/-) mice than in wild-type (WT) controls. Vasoconstriction was studied in isolated, pressurized mesenteric small arteries from WT and NCX1(SM-/-) mice and in heterozygotes with a global null mutation (NCX1(Fx/-)). Reduced NCX1 activity was manifested by a marked attenuation of responses to low extracellular Na(+) concentration, nanomolar ouabain, and SEA0400. Myogenic tone (MT, 70 mmHg) was reduced by approximately 15% in NCX1(SM-/-) arteries and, to a similar extent, by SEA0400 in WT arteries. MT was normal in arteries from NCX1(Fx/-) mice, which had normal BP. Vasoconstrictions to phenylephrine and elevated extracellular K(+) concentration were significantly reduced in NCX1(SM-/-) arteries. Because a high extracellular K(+) concentration-induced vasoconstriction involves the activation of L-type voltage-gated Ca(2+) channels (LVGCs), we measured LVGC-mediated currents and Ca(2+) sparklets in isolated mesenteric artery myocytes. Both the currents and the sparklets were significantly reduced in NCX1(SM-/-) (vs. WT or NCX1(Fx/-)) myocytes, but the voltage-dependent inactivation of LVGCs was not augmented. An acute application of SEA0400 in WT myocytes had no effect on LVGC current. The LVGC agonist, Bay K 8644, eliminated the differences in LVGC currents and Ca(2+) sparklets between NCX1(SM-/-) and control myocytes, suggesting that LVGC expression was normal in NCX1(SM-/-) myocytes. Bay K 8644 did not, however, eliminate the difference in myogenic constriction between WT and NCX1(SM-/-) arteries. We conclude that, under physiological conditions, NCX1-mediated Ca(2+) entry contributes significantly to the maintenance of MT. In NCX1(SM-/-) mouse artery myocytes, the reduced Ca(2+) entry via NCX1 may lower cytosolic Ca(2+) concentration and thereby reduce MT and BP. The reduced LVGC activity may be the consequence of a low cytosolic Ca(2+) concentration.

Low-dose ouabain constricts small arteries from ouabain-hypertensive rats: implications for sustained elevation of vascular resistance.

Prolonged ouabain administration to normal rats causes sustained blood pressure (BP) elevation. This ouabain-induced hypertension (OH) has been attributed, in part, to the narrowing of third-order resistance arteries (approximately 320 microm internal diameter) as a result of collagen deposition in the artery media. Here we describe the structural and functional properties of fourth-order mesenteric small arteries from control and OH rats, including the effect of low-dose ouabain on myogenic tone in these arteries. Systolic BP in OH rats was 138 +/- 3 versus 124 +/- 4 mmHg in controls (P < 0.01). Pressurized (70 mmHg) control and OH arteries, with only a single layer of myocytes, both had approximately 165-microm internal diameters and approximately 20-microm wall thicknesses. Even after fixation, despite vasoconstriction, the diameters and wall thicknesses did not differ between control and OH fourth-order arteries, whereas in third-order arteries, both parameters were significantly smaller in OH than in controls. Myogenic reactivity was significantly augmented in OH fourth-order arteries. Nevertheless, phenylephrine- (1 microM) and high K(+)-induced vasoconstrictions and acetylcholine-induced vasodilation were comparable in control and OH arteries. Vasoconstrictions induced by 5 microM phenylephrine and by 10 mM caffeine in Ca(2+)-free media indicated that releasable sarcoplasmic reticulum Ca(2+) stores were normal in OH arteries. Importantly, 100 nM ouabain constricted both control and OH arteries by approximately 26 microm, indicating that this response was not downregulated in OH rats. This maximal ouabain-induced constriction corresponds to a approximately 90% increase in resistance to flow in these small arteries; thus ouabain at EC(50) of approximately 0.66 nM should raise resistance by approximately 35%. We conclude that dynamic constriction in response to circulating nanomolar ouabain in small arteries likely makes a major contribution to the increased vascular tone and BP in OH rats.

Role of store-operated Ca(2+) entry in adenosine-induced vasodilatation of rat small mesenteric artery.

Store-operated Ca(2+) entry (SOCE) has recently been proposed to contribute to Ca(2+) influx in vascular smooth muscle cells (VSMCs). Adenosine is known for its protective role against hypoxia and ischemia by increasing nutrient and oxygen supply through vasodilation. This study was designed to examine the hypothesis that SOCE have a functional role in adenosine-induced vasodilation. Small mesenteric resistance arteries and mesenteric VSMCs were obtained from rats. Isometric tensions of isolated artery rings were measured by a sensitive myograph system. Laser-scanning confocal microscopy was used to determine the intracellular Ca(2+) concentration of fluo 3-loaded VSMCs. Adenosine (0.1-100 microM) relaxed artery rings that were precontracted by phenylephrine in a concentration-dependent manner. In cultured mesenteric VSMCs, passive store depletion by thapsigargin and active store depletion by phenylephrine both induced Ca(2+) influx due to SOCE. Adenosine inhibited SOCE-mediated increases in cytosolic Ca(2+) levels evoked by the emptying of the stores. In isolated artery rings, adenosine inhibited SOCE-induced contractions due to store depletion. A(2A) receptor antagonism with SCH-58261 and adenylate cyclase inhibition with SQ-22536 largely attenuated adenosine responses. The cAMP analog 8-bromo-cAMP mimicked the effects of adenosine on SOCE. Our results indicate a novel mechanism of vasodilatation by adenosine that involves regulation of SOCE through the cAMP signaling pathway due to activation of adenosine A(2A) receptors.

Sympathetic neurogenic Ca2+ signalling in rat arteries: ATP, noradrenaline and neuropeptide Y.

The sympathetic nervous system (SNS) plays an essential role in the control of total peripheral vascular resistance by controlling the contraction of small arteries. The SNS also exerts long-term trophic influences in health and disease; SNS hyperactivity accompanies most forms of human essential hypertension, obesity and heart failure. At their junctions with smooth muscle cells, the peri-arterial sympathetic nerves release ATP, noradrenaline (NA) and neuropeptide Y (NPY) onto smooth muscle cells. Confocal Ca(2+) imaging studies reveal that ATP and NA each produce unique types of postjunctional Ca(2+) signals and consequent smooth muscle cell contractions. Neurally released ATP activates postjunctional P2X(1) receptors to produce local, non-propagating Ca(2+) transients, termed 'junctional Ca(2+) transients', or 'jCaTs'. Neurally released NA binds to alpha(1)-adrenoceptors and can activate Ca(2+) waves or more uniform global changes in [Ca(2+)]. Neurally released NPY does not appear to produce Ca(2+) transients directly, but significantly modulates NA-induced Ca(2+) signalling. The neural release of ATP and NA, as judged by postjunctional Ca(2+) signals, electrical recording of excitatory junction potentials and carbon fibre amperometry to measure NA, varies markedly with the pattern of nerve activity. This probably reflects both pre- and postjunctional mechanisms, which are not yet fully understood. These phenomena, together with different temporal patterns of sympathetic nerve activity in different regional circulations, are probably an important mechanistic basis of the important selective regulation of regional vascular resistance and blood flow by the sympathetic nervous system.

A technique for simultaneous measurement of Ca2+, FRET fluorescence and force in intact mouse small arteries.

FRET (Forster resonance energy transfer)-based biosensor molecules are powerful tools to reveal specific molecular interactions in cells. Typically however, they are used in cultured cells that (inevitably) express different genes than their counterparts in intact organisms. In such cells it may be impossible to administer physiological stimuli and measure physiological outputs. Here, through the use of transgenic mice that express a FRET-based myosin light chain kinase (MLCK) biosensor molecule, we report a technique for dynamically observing activation and regulation of MLCK within the smooth muscle cells of intact, functioning small arteries, together with measurement of arterial force production and intracellular [Ca(2+)].

Evoked and spontaneous purinergic junctional Ca2+ transients (jCaTs) in rat small arteries.

Confocal microscopy of fluo-4 fluorescence in pressurized rat mesenteric small arteries subjected to low-frequency electrical field stimulation revealed Ca2+ transients in perivascular nerves and novel, spatially localized Ca2+ transients in adjacent smooth muscle cells. These muscle Ca2+ transients occur with a very brief latency to the stimulus pulse (most <3 ms). They are wider (approximately 5 micro m) and last longer (t(1/2), 145 ms) than Ca2+ sparks. They are abolished by the purinergic receptor (P2X) antagonist suramin, but they are totally unaffected by the alpha1 adrenoceptor antagonist prazosin or by capsaicin (which inhibits the function of perivascular sensory nerves). We conclude that these novel Ca2+ transients represent Ca2+ entering smooth muscle cells through P2X receptors activated by ATP released from sympathetic nerves, and we therefore call them "junctional Ca2+ transients" or jCaTs. As expected from spontaneous neurotransmitter release, jCaTs also occur spontaneously, with characteristics identical to evoked jCaTs. Visualization of sympathetic neurotransmission shows that purinergic components dominate at low frequencies of sympathetic nerve fiber activation.

Organization of Ca2+ stores in vascular smooth muscle: functional implications.

Much evidence suggests that caffeine/ryanodine (Caf/Ry)-releasable and inositol-1,4,5-trisphosphate (InsP3)-releasable Ca2+ stores in the sarcoplasmic reticulum (SR) of smooth muscles are at least partially distinct. We directly visualized SR stores in primary-cultured rat mesenteric artery myocytes with high-resolution digital imaging and the low-affinity Ca2, indicator, Furaptra (Kd = 75.6 microM). The SR appears to be a continuous tubular network. Nevertheless, SR Ca2+ stores are organized into small, separate, functionally independent compartments. Cyclopiazonic acid (CPA; inhibits SR (Ca2+ pump) and Caf (or Ry) release Ca2+ from different, spatially distinct compartments. Similar heterogeneity is seen with serotonin (acts via InsP3), which unloads only the CPA-sensitive compartments. Some of the SR ('junctional' SR; jSR) lies within 12-15 nm of the plasmalemma (PL). The jSR, the overlying PL microdomains, and the intervening, tiny volume of cytosol form junctional complexes ('PLasmERosomes'). Na+ pumps with high-ouabain-affinity alpha2 or alpha3 subunits, Na+/Ca2+ exchangers, and store-operated channels are confined to these PL microdomains, whereas Na+ pumps with low-ouabain-affinity alpha1 subunits and plasma membrane Ca2+ pumps are uniformly distributed. As a result of this organization, low-dose ouabain can selectively modulate Na+ and Ca2+ concentrations in the PLasmERosomes and jSR Ca2+ stores, and can thereby regulate Ca2+ signalling.