Effects of Deoxycholylglycine, a Conjugated Secondary Bile Acid, on Myogenic Tone and Agonist-Induced Contraction in Rat Resistance Arteries

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.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.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

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°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 α1-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 Y1 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 (10−11–10−7M)-induced vasoconstriction, which is caused by both enhanced spontaneous NE release and direct effects on smooth muscle. Ouabain (<10−7M) 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 Ca2+ concentration ([Ca2+]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 [Ca2+]i, MLCK activity, and diameter in arteries, uncomplicated by systemic effects. Transgenic mice that express, in smooth muscle, a Ca2+/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 ∼90 mmHg, femoral arteries were constricted to 65% of their passive diameter, MLCK fractional activation was 0.14, and [Ca2+]i was 131 nM. Phenylephrine (300 ng/g wt iv) elevated mean BP transiently to ∼110 mmHg, decreased heart rate, increased femoral artery [Ca2+]i to 244 nM and fractional MLCK activation to 0.24, and decreased artery diameter by 23%. In comparison, local application of 1.0 μM phenylephrine raised [Ca2+]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 [Ca2+]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 [Ca2+]i of 29 nM between two groups of 12 mice with a significance of P < 0.05

Effect of DCG on agonist-induced vasoconstriction.

<p>(A) At 70 mmHg, the arteries developed spontaneous vasoconstriction that was further augmented by adding ANG II (0.1 µM). DCG attenuated ANG II-induced vasoconstriction. (n = 5 arteries). (B) An example tracing of the effect of DCG on ANG II-induced vasoconstriction. (C) At 70 mmHg, the arteries developed spontaneous vasoconstriction that was further augmented by adding PE (1 µM). DCG attenuated PE-induced vasoconstriction. (n = 12 arteries). (D) DCG (100 µM) did not alter 80 mM KCl-induced vasoconstriction. (n = 3 arteries). Asterisks indicate significant differences when compared to ANG II and PE alone.</p

Effect of DCG and ACh on MT in rat 4^th-order mesenteric arteries.

<p>(A) DCG induces concentration-dependent reduction of MT in endothelium-intact and -denuded arteries. Asterisks indicate significance when compared to the baseline for both preparations. (B) DCG (100 µM) reduced MT in endothelium-intact and -denuded arteries. This effect was reversed by washing with PSS; (WO-Wash out). Asterisks indicate significance when compared to DCG (100 µM)-mediated response in respective arterial preparations. (C) ACh induced concentration-dependent reduction of MT. There was no effect in vehicle-treated arteries. (D) Addition of 10 µM ACh had no effect on endothelium-denuded preparations while endothelium-intact preparation achieved diameters similar to passive diameter. Asterisks indicate significance when compared to untreated endothelium-intact arteries. The dashed-line represents passive diameter (PD). PD is the lumen diameter when VSM is completely inactive. (n = 4–5 arteries in each group).</p

High Vascular Tone of Mouse Femoral Arteries <i>In Vivo</i> Is Determined by Sympathetic Nerve Activity Via α_1A- and α_1D-Adrenoceptor Subtypes

<div><p>Background and purpose</p><p>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.</p><p>Experimental approach</p><p>Diameter and intracellular calcium ([Ca²⁺]_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 [Ca²⁺]_i,.</p><p>Key results</p><p>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 P_2X 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 (α_1A-selective antagonist) and BMY7378 (α_1D-selective) decreased tone by 29% and 26%, respectively, and reduced [Ca²⁺]_i. Chloroethylclonidine (1 µM preferential for α_1B-) had no effect. Abolition of sympathetic nerve activity (hexamethonium, i.p.) reduced basal tone by 90%.</p><p>Conclusion and Implications</p><p>Tone of mouse femoral arteries in vivo is almost entirely sympathetic in origin. Activation of α_1A- and α_1D-adrenoceptors elevates [Ca²⁺]_i and accounts for at least 55% of the tone.</p></div

DCG reduces VSM Ca²⁺ in 4^th-order mesenteric arteries from rats with MT.

<p>Ca²⁺ fluorescence was measured in Fluo-2-loaded arteries before and after incubating with DCG 100 µM for 5 minutes. DCG reduced the arterial VSM Ca²⁺ fluorescence by ∼50%. (n = 3 arteries in each group).</p

Second-round PCR Primer Sequences.

<p>bp-base pair.</p