Notch transcriptional control of vascular smooth muscle regulatory gene expression and function.

Notch receptors and ligands mediate heterotypic cell signaling that is required for normal vascular development. Dysregulation of select Notch receptors in mouse vascular smooth muscle (VSM) and in genetic human syndromes causes functional impairment in some regional circulations, the mechanistic basis of which is undefined. In this study, we used a dominant-negative Mastermind-like (DNMAML1) to block signaling through all Notch receptors specifically in VSM to more broadly test a functional role for this pathway in vivo. Mutant DNMAML1-expressing mice exhibited blunted blood pressure responses to vasoconstrictors, and their aortic, femoral, and mesenteric arteries had reduced contractile responses to agonists and depolarization in vitro. The mutant arteries had significant and specific reduction in the expression and activity of myosin light chain kinase (MLCK), a primary regulator of VSM force production. Conversely, activated Notch signaling in VSM cells induced endogenous MLCK transcript levels. We identified MLCK as a direct target of activated Notch receptor as demonstrated by an evolutionarily conserved Notch-responsive element within the MLCK promoter that binds the Notch receptor complex and is required for transcriptional activity. We conclude that Notch signaling through the transcriptional control of key regulatory proteins is required for contractile responses of mature VSM. Genetic or pharmacological manipulation of Notch signaling is a potential strategy for modulating arterial function in human disease.

Increased arterial smooth muscle Ca2+ signaling, vasoconstriction, and myogenic reactivity in Milan hypertensive rats.

The Milan hypertensive strain (MHS) rats are a genetic model of hypertension with adducin gene polymorphisms linked to enhanced renal tubular Na(+) reabsorption. Recently we demonstrated that Ca(2+) signaling is augmented in freshly isolated mesenteric artery myocytes from MHS rats. This is associated with greatly enhanced expression of Na(+)/Ca(2+) exchanger-1 (NCX1), C-type transient receptor potential (TRPC6) protein, and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) compared with arteries from Milan normotensive strain (MNS) rats. Here, we test the hypothesis that the enhanced Ca(2+) signaling in MHS arterial smooth muscle is directly reflected in augmented vasoconstriction [myogenic and phenylephrine (PE)-evoked responses] in isolated mesenteric small arteries. Systolic blood pressure was higher in MHS (145 ± 1 mmHg) than in MNS (112 ± 1 mmHg; P < 0.001; n = 16 each) rats. Pressurized mesenteric resistance arteries from MHS rats had significantly augmented myogenic tone and reactivity and enhanced constriction to low-dose (1-100 nM) PE. Isolated MHS arterial myocytes exhibited approximately twofold increased peak Ca(2+) signals in response to 5 µM PE or ATP in the absence and presence of extracellular Ca(2+). These augmented responses are consistent with increased vasoconstrictor-evoked sarcoplasmic reticulum (SR) Ca(2+) release and increased Ca(2+) entry, respectively. The increased SR Ca(2+) release correlates with a doubling of inositol 1,4,5-trisphosphate receptor type 1 and tripling of SERCA2 expression. Pressurized MHS arteries also exhibited a ∼70% increase in 100 nM ouabain-induced vasoconstriction compared with MNS arteries. These functional alterations reveal that, in a genetic model of hypertension linked to renal dysfunction, multiple mechanisms within the arterial myocytes contribute to enhanced Ca(2+) signaling and myogenic and vasoconstrictor-induced arterial constriction. MHS rats have elevated plasma levels of endogenous ouabain, which may initiate the protein upregulation and enhanced Ca(2+) signaling. These molecular and functional changes provide a mechanism for the increased peripheral vascular resistance (whole body autoregulation) that underlies the sustained hypertension.

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.

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.

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+)].

Decreased activity of the smooth muscle Na+/Ca2+ exchanger impairs arteriolar myogenic reactivity.

Arteriolar myogenic vasoconstriction occurs when stretch or increased membrane tension leads to smooth muscle cell (SMC) depolarization and opening of voltage-gated Ca(2+) channels. While the mechanism underlying the depolarization is uncertain a role for non-selective cation channels has been demonstrated. As such channels may be expected to pass Na(+), we hypothesized that reverse mode Na(+)/Ca(2+) exchange (NCX) may act to remove Na(+) and in addition play a role in myogenic signalling through coupled Ca(2+) entry. Further, reverse (Ca(2+) entry) mode function of the NCX is favoured by the membrane potential found in myogenically active arterioles. All experiments were performed on isolated rat cremaster muscle first order arterioles (passive diameter approximately 150 mum) which were pressurized in the absence of intraluminal flow. Reduction of extracellular Na(+) to promote reverse-mode NCX activity caused significant, concentration-dependent vasoconstriction and increased intracellular Ca(2+). This vasoconstriction was attenuated by the NCX inhibitors KB-R7943 and SEA 04000. Western blotting confirmed the existence of NCX protein while real-time PCR studies demonstrated that the major isoform expressed in the arteriolar wall was NCX1. Oligonucleotide knockdown (24 and 36 h) of NCX inhibited the vasoconstrictor response to reduced extracellular Na(+) while also impairing both steady-state myogenic responses (as shown by pressure-diameter relationships) and acute reactivity to a 50 to 120 mmHg pressure step. The data are consistent with reverse mode activity of the NCX in arterioles and a contribution of this exchanger to myogenic vasoconstriction.

High vascular tone of mouse femoral arteries in vivo is determined by sympathetic nerve activity via α1A- and α1D-adrenoceptor subtypes.

BACKGROUND AND PURPOSE: Determining the role of vascular receptors in vivo is difficult and not readily accomplished by systemic application of antagonists or genetic manipulations. Here we used intravital microscopy to measure the contributions of sympathetic receptors, particularly α1-adrenoceptor subtypes, to contractile activation of femoral artery in vivo. EXPERIMENTAL APPROACH: Diameter and intracellular calcium ([Ca(2+)]i) in femoral arteries were determined by intravital fluorescence microscopy in mice expressing a Myosin Light Chain Kinase (MLCK) based calcium-calmodulin biosensor. Pharmacological agents were applied locally to the femoral artery to determine the contributions of vascular receptors to tonic contraction and [Ca(2+)]i,. KEY RESULTS: In the anesthetized animal, femoral arteries were constricted to a diameter equal to 54% of their passive diameter (i.e. tone = 46%). Of this total basal tone, 16% was blocked by RS79948 (0.1 µM) and thus attributable to α2-adrenoceptors. A further 46% was blocked by prazosin (0.1 µM) and thus attributable to α1-adrenoceptors. Blockade of P2X and NPY1 receptors with suramin (0.5 mM) and BIBP3226 (1.0 µM) respectively, reduced tone by a further 22%, leaving 16% of basal tone unaffected at these concentrations of antagonists. Application of RS100329 (α1A-selective antagonist) and BMY7378 (α1D-selective) decreased tone by 29% and 26%, respectively, and reduced [Ca(2+)]i. Chloroethylclonidine (1 µM preferential for α1B-) had no effect. Abolition of sympathetic nerve activity (hexamethonium, i.p.) reduced basal tone by 90%. CONCLUSION AND IMPLICATIONS: Tone of mouse femoral arteries in vivo is almost entirely sympathetic in origin. Activation of α1A- and α1D-adrenoceptors elevates [Ca(2+)]i and accounts for at least 55% of the tone.

Effects of deoxycholylglycine, a conjugated secondary bile acid, on myogenic tone and agonist-induced contraction in rat resistance arteries.

BACKGROUND: Bile acids (BAs) regulate cardiovascular function via diverse mechanisms. Although in both health and disease serum glycine-conjugated BAs are more abundant than taurine-conjugated BAs, their effects on myogenic tone (MT), a key determinant of systemic vascular resistance (SVR), have not been examined. METHODOLOGY/PRINCIPAL FINDINGS: Fourth-order mesenteric arteries (170-250 µm) isolated from Sprague-Dawley rats were pressurized at 70 mmHg and allowed to develop spontaneous constriction, i.e., MT. Deoxycholylglycine (DCG; 0.1-100 µM), a glycine-conjugated major secondary BA, induced reversible, concentration-dependent reduction of MT that was similar in endothelium-intact and -denuded arteries. DCG reduced the myogenic response to stepwise increase in pressure (20 to 100 mmHg). Neither atropine nor the combination of L-NAME (a NOS inhibitor) plus indomethacin altered DCG-mediated reduction of MT. K(+) channel blockade with glibenclamide (K(ATP)), 4-aminopyradine (K(V)), BaCl(2) (K(IR)) or tetraethylammonium (TEA, K(Ca)) were also ineffective. In Fluo-2-loaded arteries, DCG markedly reduced vascular smooth muscle cell (VSM) Ca(2+) fluorescence (∼50%). In arteries incubated with DCG, physiological salt solution (PSS) with high Ca(2+) (4 mM) restored myogenic response. DCG reduced vascular tone and VSM cytoplasmic Ca(2+) responses (∼50%) of phenylephrine (PE)- and Ang II-treated arteries, but did not affect KCl-induced vasoconstriction. CONCLUSION: In rat mesenteric resistance arteries DCG reduces pressure- and agonist-induced vasoconstriction and VSM cytoplasmic Ca(2+) responses, independent of muscarinic receptor, NO or K(+) channel activation. We conclude that BAs alter vasomotor responses, an effect favoring reduced SVR. These findings are likely pertinent to vascular dysfunction in cirrhosis and other conditions associated with elevated serum BAs.