Rat Small Mesenteric Arteries Research Articles

The α2 isoform of the Na, K‐pump is involved in synchronization of smooth muscle cells in the arterial wall.

We have hypothesized that the ouabain‐sensitive Na, K‐pump can regulate smooth muscle gap junction function. To test this hypothesis and determine the relevant isoform we investigated the effect of downregulation of the α2 isoform of Na, K‐pump.We transfected cultured smooth muscle cells (SMCs) and rat small mesenteric arteries with siRNA against the α2 isoform of Na, K‐pump and with eGFP siRNA, as a control. Paired SMCs were used as a model for electrical coupling by measuring membrane capacitance and synchronized SMC activity was assessed by measuring isometric force.Pairs of SMCs had a capacitance of 90.4±9.4pF, n=7 twice that of single cells. SMCs were uncoupled when the Na, K‐pump was inhibited by 10 μM ouabain (46.1±7.2pF). β‐glycyrrhetinic acid (βGA) had no additive effect (41.9±4.9pF). Downregulation of the α2 Na, K‐pump significantly reduced the capacitance of cell pairs (33.4±4.1pF) and made them insensitive to ouabain (34.1±4.2pF). βGA uncoupled residual coupling (28.3±3.0pF). Arteries downregulated for the Na, K‐pump had significant reduced amplitude of noradrenalin‐induced vasomotion. This is consistent with the suggested role of gap junction for synchronization of SMCs in the vascular wall.We demonstrate the importance of α2 isofom of the Na, K‐pump for intercellular coupling in the vascular wall and its involvement in the regulation of vasomotion.

Intracellular Ca 2+ Silences L-Type Ca 2+ Channels in Mesenteric Veins

Rationale : Calcium channel blockers (CCBs) exert their antihypertensive effect by reducing cardiac afterload but not preload, suggesting that Ca 2+ influx through L-type Ca 2+ channels (LTCC) mediates arterial but not venous tone. Objective : The object of this study was to resolve the mechanism of venous resistance to CCBs. Methods and Results : We compared the sensitivity of depolarization (KCl)-induced constriction of rat small mesenteric arteries (MAs) and veins (MVs) to the dilator effect of CCBs. Initial findings confirmed that nifedipine progressively dilated depolarization-induced constrictions in MAs but not MVs. However, Western blots showed a similar expression of the α 1C pore-forming subunit of the LTCC in both vessels. Patch-clamp studies revealed a similar density of whole-cell Ca 2+ channel current between single smooth muscle cells (SMCs) of MAs and MVs. Based on these findings, we hypothesized that LTCCs are expressed but “silenced” by intracellular Ca 2+ in venous SMCs. After depletion of intracellular Ca 2+ stores by the SERCA pump inhibitor thapsigargin, depolarization-induced constrictions in MVs were blocked 80% by nifedipine suggesting restoration of Ca 2+ influx through LTCCs. Similarly, KCl-induced constrictions were sensitive to block by nifedipine after depletion of intracellular Ca 2+ stores by caffeine, ryanodine, or 2-aminoethoxydiphenyl borate. Cell-attached patch recordings of unitary LTCC currents confirmed rare channel openings during depolarization of venous compared to arterial SMCs, but chelating intracellular Ca 2+ significantly increased the open-state probability of venous LTCCs. Conclusions : We report that intracellular Ca 2+ inactivates LTCCs in venous SMCs to confer venous resistance to CCB-induced dilation, a fundamental drug property that was previously unexplained.

Differential recruitment of mechanisms for myogenic responses according to luminal pressure and arterial types

Mechanosensitive nonselective cation channels (NSC(ms)), protein kinase C (PKC), and Rho kinase (ROCK) are suggested as underlying mechanisms for the myogenic contractile response (MR) to luminal pressure (P(lum)). Here we compared relative contributions from these mechanisms using pharmacological inhibitors in rabbit middle cerebral (RbCA), rat middle cerebral (RtCA), rat femoral (RtFA), and rat mesenteric (RtMA) small arteries. Inner diameters of pressurized arteries under various P(lum) were video-analyzed. 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 10 microM) was used as a blocker of NSC(ms). In general, RbCA and RtCA showed higher P(lum) sensitivity of MR than RtFA and RtMA. Ten micromolars of DIDS commonly decreased MRs more effectively at low P(lum) (40-60 mmHg) in all tested arteries except RtCA. In RbCA, PKC inhibitors (100 nM of Go6976 or Go6983) decreased the MR at relatively high P(lum) (80-100 mmHg) whereas ROCK inhibitor (Y-27632, 1 microM) showed a P(lum)-independent inhibition. In RtMA and RtCA, PKC inhibitors (Go6976 and Go6983) had no significant effect whereas Y-27632 generally inhibited the MR. In RtFA, neither PKC inhibitor nor Y-27632 alone affected MRs. Interestingly, in the presence of 10 microM DIDS, Go6983 and Y-27632 decreased the MR of RtFA. In RtMA, it was notable that the MR decreased spontaneously on repeated protocol of P(lum) increase, and the 'run-down' could be effective reversed by maxi-K(+) channel blocker (tetraethylammonium or iberiotoxin). In summary, our study shows the variability of MRs according to the arterial types in terms of their pressure sensitivity and underlying mechanisms that are recruited according to P(lum).

Vasorelaxant activities of the putative endocannabinoid virodhamine in rat isolated small mesenteric artery.

Virodhamine is a recently identified novel endocannabinoid. Cannabinoids may evoke vasorelaxation through novel receptors in the vasculature and/or through release of vasodilator peptides from sensory nerve endings. Virodhamine induced endothelium-dependent relaxation in the rat isolated small mesenteric artery mounted in a myograph and precontracted with methoxamine. Desensitization of vanilloid receptors by capsaicin did not affect relaxation responses to virodhamine. The CB(1) receptor antagonist SR 141716A (3 microM), but not the more CB(1)-selective blocker AM 251 (1 microM), attenuated the response, while two CB(2) receptor antagonists, SR 144528 (1 microM) and AM 630 (10 microM), had no effect. The novel antagonist for the putative endothelial 'abnormal-cannabidiol receptor', O-1918 (30 microM), inhibited virodhamine relaxations. Hence virodhamine may activate this novel receptor, which might also recognize SR 141716A. Inhibition of nitric oxide synthase (L-NAME 300 microM) did not affect relaxation to virodhamine but the responses were markedly reduced when tone was induced with 60 mM KCl, suggesting a role for the activation of K(+) channels. The Ca(2+)-activated K(+) channel (K(Ca)) blockers, apamin (50 nM) and charybdotoxin (50 nM), inhibited virodhamine vasorelaxation. Combination of these blockers with SR 141716A (3 microM) caused no further inhibition. It was concluded that virodhamine relaxes the rat small mesenteric artery by endothelium-dependent activation of K(Ca), perhaps via the putative abnormal-cannabidiol receptor.

Mechanism of the vasodilator effect of Euxanthone in rat small mesenteric arteries

In the present work we investigated the mechanism involved in the vasodilator effect induced by euxanthone in rat small mesenteric arteries. We observed that euxanthone induced concentration-dependent vasodilatation in arteries by a mechanism independent on the release of endothelial factors, such as nitric oxide (NO) and cyclooxygenase-derived factors. In addition our results also suggest that euxanthone induced its vasodilator effect through inhibition of calcium-sensitive mechanisms activated by protein kinase C, rather than by inhibition of contractions dependent on the release of the intracellular calcium stores or by inhibition of voltage-operated calcium channels.

Functional Role of Vanilloid Transient Receptor Potential 4-Canonical Transient Receptor Potential 1 Complex in Flow-Induced Ca 2+ Influx

The present study is aimed at investigating the interaction of TRPV4 with TRPC1 and the functional role of such an interaction in flow-induced Ca(2+) influx. Hemodynamic blood flow is an important physiological factor that modulates vascular tone. One critical early event in this process is a cytosolic Ca(2+) ([Ca(2+)](i)) rise in endothelial cells in response to flow. With the use of fluorescence resonance energy transfer, coimmunoprecipitation, and subcellular colocalization methods, it was found that TRPC1 interacts physically with TRPV4 to form a complex. In functional studies, flow elicited a transient [Ca(2+)](i) increase in TRPV4-expressing human embryonic kidney (HEK) 293 cells. Coexpression of TRPC1 with TRPV4 markedly prolonged this [Ca(2+)](i) transient; it also enabled this [Ca(2+)](i) transient to be negatively modulated by protein kinase G. Furthermore, this flow-induced [Ca(2+)](i) increase was markedly inhibited by anti-TRPC1-blocking antibody T1E3 and a dominant-negative construct TRPC1 Delta 567-793 in TRPV4-C1-coexpressing HEK cells and human umbilical vein endothelial cells. T1E3 also inhibited flow-induced vascular dilation in isolated rat small mesenteric artery segments. This study shows that TRPC1 interacts physically with TRPV4 to form a complex, and this TRPV4-C1 complex may mediate flow-induced Ca(2+) influx in vascular endothelial cells. The association of TRPC1 with TRPV4 prolongs the flow-induced [Ca(2+)](i) transient, and it also enables this [Ca(2+)](i) transient to be negatively modulated by protein kinase G. This TRPV4-C1 complex plays a key role in flow-induced endothelial Ca(2+) influx.

Desensitization of endothelial P2Y1 receptors by PKC‐dependent mechanisms in pressurized rat small mesenteric arteries

Extracellular nucleotides play a crucial role in the regulation of vascular tone and blood flow. Stimulation of endothelial cell P2Y1 receptors evokes concentration-dependent full dilatation of resistance arteries. However, this GPCR can desensitize upon prolonged exposure to the agonist. Our aim was to determine the extent and nature of P2Y1 desensitization in isolated and pressurized rat small mesenteric arteries. The non-hydrolyzable selective P2Y1 agonist ADPbetaS (3 microM) was perfused through the lumen of arteries pressurized to 70 mmHg. Changes in arterial diameter and endothelial cell [Ca(2+)](i) were obtained in the presence and absence of inhibitors of protein kinase C (PKC). ADPbetaS evoked rapid dilatation to the maximum arterial diameter but faded over time to a much-reduced plateau closer to 35% dilatation. This appeared to be due to desensitization of the P2Y1 receptor, as subsequent endothelium-dependent dilatation to acetylcholine (1 microM) remained unaffected. Luminal treatment with the PKC inhibitors BIS-I (1 microM) or BIS-VIII (1 microM) tended to augment concentration-dependent dilatation to ADPbetaS (0.1-3 microM) and prevented desensitization. Another PKC inhibitor, Gö 6976 (1 microM), was less effective in preventing desensitization. Measurements of endothelial cell [Ca(2+)](i) in pressurized arteries confirmed the P2Y1 receptor but not M(3) muscarinic receptor desensitization. These data demonstrate for the first time the involvement of PKC in the desensitization of endothelial P2Y1 receptors in pressurized rat mesenteric arteries, which may have important implications in the control of blood flow by circulating nucleotides.

Maintenance of adrenergic vascular tone by MMP transactivation of the EGFR requires PI3K and mitochondrial ATP synthesis

G-protein-coupled receptors (GPCRs) modulate vascular tone, at least in part, via matrix metalloproteinase (MMP) transactivation of the epidermal growth factor receptor (EGFR). We previously have identified novel signalling pathways downstream of the EGFR suggestive of mitogen-activated protein kinase and mitochondrial redox control of vascular tone. In the present study, we examined whether MMP modulation of vascular tone involves phosphoinositide 3-kinase (PI3K) and mitochondrial ATP synthesis. To determine whether PI3K is required for the maintenance of adrenergic vascular tone, we first constricted rat small mesenteric arteries with phenylephrine (PE) and then perfused with PI3K inhibitors, LY294002 and wortmannin, both of which produced a dose-dependent vasodilatation. Next, to investigate whether MMPs modulate PI3K activity, we cultured rat aortic vascular smooth muscle cells (VSMCs) and stimulated them with GPCR agonists such as PE and angiotensin II. Inhibition of MMPs (by GM6001) or EGFR (by AG1478) or suppressing the expression of MMP-2 or MMP-7 or the EGFR by small interfering RNA blunted the PI3K phosphorylation of Akt induced by PE. Further, in VSMCs, PI3K inhibitors reduced the PE-induced increase in ATP synthesis and glucose transporter-4 translocation, an effect that was also observed with MMP and the EGFR inhibitors. Further, the PE-induced increase in ATP synthesis activated MMP-7 by mechanisms involving purinergic (P2X) receptors and calcium. These data suggest that the maintenance of adrenergic vascular tone by the MMP-EGFR pathway requires PI3K activation and ATP synthesis. Further, our data support the view that elevated levels of GPCR agonists exaggerate the MMP transactivation of EGFR response and contribute to enhanced vascular tone and development of cardiovascular disease such as hypertension.

Mechanisms underlying the vasorelaxant effect induced by Anacardium occidentale L. leaf fraction in rat small resistance mesenteric arteries

Anacardium occidentale L. (A. occidentale), belonging to Anacardiaceae family, has been documented as traditional plant for the treatment of diabetes and hypertension [1]. Four extracts of A. occidentale were used for this study, namely AOL1 (from cyclohexane), AOL2 (from CH2Cl2), AOL3 (from EtOAc) and AOL4 (from MeOH). The most potent antiradical reactivity was observed in AOL4 with 2023 TE/g of extract compared to chlorogenic acid (2976 TE/g of compound) as positive standard. In the other hand, our data showed a significant inhibitory effect of EtOAc and MeOH extracts on BSA glycoxidation measured in terms of advanced glycation end products (AGEs). In isolated mesenteric artery rings precontracted with phenylephrine [2], AOL1, AOL2, AOL3, and AOL4 induced a concentration-dependant relaxation respectively with IC50 values of 120µg/ml, 100µg/ml, 70µg/ml and 70µg/ml. Exposure of Eahy cells to high glucose for 7 days significantly (p

Role of store-operated Ca2+ 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.

Flow Dilation in Rat Small Mesenteric Arteries is Mediated by Hydrogen Peroxide Generated from CYP Epoxygenases and Xanthine Oxidase

We have previously demonstrated that hydrogen peroxide (H2O2) mediates flow-induced vascular dilation in rat small mesenteric arteries. In the present study, the source of this flow-induced H2O2 was explored. The arteries were pressurized to 50 mm Hg and preconstricted with phenylephrine. Intraluminal flow reversed the effect of phenylephrine, resulting in vascular dilation. Cytochrome P450 (CYP) inhibitors N-methylsulfonyl-6-(2-proparglyoxyphenyl) hexanoic acid (MS-PPOH, 10 �M) and miconazole (30 �M) reduced the magnitude of peak flow dilation by ~20%-30%, and reduced the duration of dilatory response by ~70-80%. Nevertheless, sulphaphenazole (10 �M), a selective inhibitor of CYP 2C9, had no effect neither on the peak flow dilation nor the duration of dilatory response. Oxypurinol (100 �M), an inhibitor of xanthine oxidase, attenuated the duration of dilatory response by ~60% but exerted no effect on the magnitude of peak flow dilation. Cyclosporin A (2 �M), an inhibitor for mitochondrial permeability transition pore, MitoQ (300 nM), a mitochondria-targeted antioxidant, and apocynin (1 mM), a NADPH oxidase inhibitor, had no effect neither on the magnitude of peak flow dilation nor the duration of dilatory response. To further confirm the role of CYP in flow-induced H2O2 production, a fluorescent probe CM-H2DCFDA was used to monitor the production of H2O2 in the primary endothelial cells isolated from rat small mesenteric arteries. The results showed that flow-induced H2O2 production was markedly reduced in MS-PPOH and miconazole pretreated endothelial cells. Taken together, our results suggest that, during flow dilation, H2O2 is generated from CYP epoxygenases and xanthine oxidase.

Acute TiO2 exposure does not alter endothelial function in rat small mesenteric arteries

Owing to their high surface area to volume ratio, nanomaterials have potential benefits in industry, medicine, and household products. Although the use of nanomaterials is rapidly growing the physiological consequences of acute and chronic exposure have not been established. Indeed, short‐term (24 hr) nanomaterial exposure has been shown to increase reactive oxygen species generation (ROS) in epithelial cells in culture. An increase in ROS production within the vasculature could potentially elicit a decrease in NO bioavailability. Previous work has suggested that this increase in ROS production may involve cellular internalization. However, we hypothesized that nanomaterials interact with the endothelial cell surface resulting in an impaired endothelial‐dependent vasodilation. To this end, isolated, pressurized small mesenteric arteries (100 ‐ 250 μm) from Wistar rats were pre‐constricted with Phenylephrine (PE; 10−7 ‐ 10−6 M) and administered acetylcholine (ACh; 10−8 ‐ 10−6 M) in the presence of either vehicle or 0.05% v/v titanium dioxide (TiO2; 1 hr). ACh administration induced a dose‐dependent vasodilation. There were no statistically significant differences in the vasodilatory response to ACh between vehicle and TiO2 treated arteries. These results suggest that acute TiO2 exposure does not alter ACh induced vasodilation in small rat mesenteric arteries.

Intracellular calcium silences L‐Type Ca2+ channels in rat small mesenteric veins

Calcium channel blockers (CCBs) exert their antihypertensive effect by reducing cardiac afterload but not preload, suggesting that L‐type Ca2+ (CaL) channels contribute to arterial tone but are silent in resistance veins. Using rat small mesenteric arteries (MA) and veins (MV) perfused at their physiological pressures, we confirmed that the CCB nifedipine prevents KCl‐induced constriction of MA but not MV. We hypothesized that MVs rely on intracellular Ca2+ ([Ca]i) rather than CaL channels for constriction and that [Ca]i silences CaL channels in MVs. Indeed, the SERCA inhibitor, thapsigargin (1 μM), constricted MV but not MA (88±2% vs 3±3% relative to 60mM KCl constriction, respectively), suggesting that SR Ca2+ may fuel MV constriction. When [Ca]i was depleted by thapsigargin and extracellular Ca2+ ([Ca]o) omitted, KCl failed to constrict MVs. After depletion of [Ca]i by thapsigargin in MV and subsequent restoration of 1.5 mM [Ca]o, KCl (60 mM) contractions were partially (61±1%) restored but these contractions were blocked (61%) by 1 μM nifedipine. These data suggest: 1) a link between SR Ca2+ and constriction in MV and 2) that depletion of [Ca]i in MV disinhibits CaL channels, restoring CaL‐mediated Ca2+ influx as a source of venoconstriction. Thus, drugs that selectively deplete venous [Ca]i may represent novel therapies for the treatment of hypertension and other diseases that exhibit elevated venous tone.

Vascular Dilation Responses of Rat Small Mesenteric Arteries at High Intravascular Pressure in Spontaneously Hypertensive Rats

Hypertension is associated with remodeling and mechanical alterations of resistance arteries. Numerous studies have investigated the mechanical and morphometric properties of small arteries obtained from hypertensive animals and humans. However, the functional properties of resistance arteries from normotensive and hypertensive subjects have only been examined under normotensive conditions. The objective of the present study was to evaluate the dilation responses of small mesenteric arteries (SMA) from spontaneously hypertensive rats (SHR) at various levels of intraluminal pressure. SMA segments from Wistar Kyoto (WKY) rats and SHR were pressurized using pressure myography. Endothelium-dependent and -independent dilation responses of the SMA were examined under 3 different intravascular pressures (50, 80 and 120 mmHg). Endothelium-dependent dilation was evaluated by measuring vasodilator responses to increasing doses of acetylcholine or increases in intraluminal flow rate. Endothelium-independent vasodilator function was examined by using sodium nitroprusside. The results indicate that both endothelium-dependent and -independent dilation responses of SMA from WKY progressively decrease with increased intravascular pressure. In contrast, all dilatation responses of the SMA from SHR were enhanced at higher intraluminal pressures. These findings of differential sensitivity to luminal pressure should be considered during in vitro examination of vessels from normotensive and hypertensive subjects.

‘Entourage’ effects of N‐palmitoylethanolamide and N‐oleoylethanolamide on vasorelaxation to anandamide occur through TRPV1 receptors

The endocannabinoid N-arachidonoylethanolamide (anandamide) is co-synthesized with other N-acylethanolamides, namely N-palmitoylethanolamide (PEA) and N-oleoylethanolamide (OEA), which have been shown to potentiate anandamide responses (so-called 'entourage effects') in non-vascular tissues. It remains unclear whether such interactions occur in the circulation. In rat isolated small mesenteric arteries, the effects of PEA and OEA on relaxation to anandamide and tissue contents of the N-acylethanolamides were examined under myographic conditions. Anandamide-induced relaxation was potentiated by pretreatment with PEA (10 microM) or OEA (1 microM), or in combination. The potentiation by PEA and OEA was endothelium-independent and abolished by treatment with capsaicin (10 microM), which desensitizes the transient receptor potential vanilloid type 1 (TRPV1) receptor system, or by the TRPV1 receptor antagonist, N-(3-methoxyphenyl)-4-chlorocinnamide (SB366791) (2 microM). It was also observed at molar ratios of anandamide and PEA (or OEA) similar to those found in mesenteric arteries. PEA and inhibition of anandamide hydrolysis by 3'-carbamoyl-biphenyl-3-yl-cyclohexylcarbamate (URB597) (1 microM) additively potentiated anandamide responses. On the other hand, PEA and OEA also induced vasorelaxation per se (rank order of potency: anandamide>OEA>PEA), but relaxation to the three N-acylethanolamides displayed different sensitivity to treatment with capsaicin, SB366791 and URB597. For example, relaxations to anandamide and OEA, but not PEA, were attenuated by both capsaicin and SB366791. This study shows that PEA and OEA potentiate relaxant responses to anandamide through TRPV1 receptors in rat small mesenteric arteries. The congeners also induce vasorelaxation per se, suggesting a function for the N-acylethanolamides in vascular control.

Myosin phosphorylation triggers actin polymerization in vascular smooth muscle

A variety of contractile stimuli increases actin polymerization, which is essential for smooth muscle contraction. However, the mechanism(s) of actin polymerization associated with smooth muscle contraction is not fully understood. We tested the hypothesis that phosphorylated myosin triggers actin polymerization. The present study was conducted in isolated intact or beta-escin-permeabilized rat small mesenteric arteries. Reductions in the 20-kDa myosin regulatory light chain (MLC20) phosphorylation were achieved by inhibiting MLC kinase with ML-7. Increases in MLC20 phosphorylation were achieved by inhibiting myosin light chain phosphatase with microcystin. Isometric force, the degree of actin polymerization as indicated by the F-actin-to-G-actin ratio, and MLC20 phosphorylation were determined. Reductions in MLC20 phosphorylation were associated with a decreased force development and actin polymerization. Increased MLC20 phosphorylation was associated with an increased force generation and actin polymerization. We also found that a heptapeptide that mimics the actin-binding motif of myosin II enhanced microcystin-induced force generation and actin polymerization without affecting MLC20 phosphorylation in beta-escin-permeabilized vessels. Collectively, our data demonstrate that MLC20 phosphorylation is capable of triggering actin polymerization. We further suggest that the binding of myosin to actin triggers actin polymerization and enhances the force development in arterial smooth muscle.

Oleanolic acid induces relaxation and calcium‐independent release of endothelium‐derived nitric oxide

The present study investigated the mechanisms by which oleanolic acid, a component of olive oil, increases release of nitric oxide (NO). Measurements of isometric tension, NO concentration, or endothelial cell calcium were made in rat isolated mesenteric arteries. Immunoblotting for endothelial NOS (eNOS) and Akt kinase were performed in primary cultures of human umbilical vein endothelial cells (HUVECs). Oleanolic acid (3-30 microM) evoked endothelium-dependent relaxations in noradrenaline-contracted rat superior and small mesenteric arteries. In rat superior mesenteric arteries, oleanolic acid induced simultaneous increases in NO concentration and relaxation, and these responses were inhibited by an inhibitor of NOS, asymmetric dimethyl-L-arginine (300 microM) and by the NO scavenger, oxyhaemoglobin (10 microM). Oleanolic acid-evoked NO increases were not reduced in Ca(2+)-free solution and in the presence of an inhibitor of endoplasmic reticulum calcium-ATPase, thapsigargin (1 microM). Oleanolic acid evoked relaxation without changes in endothelial cell calcium, but decreased smooth muscle calcium in arterial segments. Oleanolic acid failed to increase calcium in HUVECs, but increased time-dependently phosphorylation of Akt kinase at Serine(473) (Akt-Ser(473)) and eNOS at Serine(1177) (eNOS-Ser(1177)), which was attenuated by inhibitors of phosphoinositide-3-kinase. This study provides direct evidence that a component of olive oil, oleanolic acid, activated endothelium-dependent release of NO and decreased smooth muscle cell calcium followed by relaxation. The oleanolic acid-evoked endothelium-derived NO release was independent of endothelial cell calcium and involved phosphoinositide-3-kinase-dependent phosphorylation of Akt-Ser(473) followed by phosphorylation of eNOS-Ser(1177).

Vascular smooth muscle dysfunction induced by monomethylarsonous acid (MMA III): A contributing factor to arsenic-associated cardiovascular diseases

While arsenic in drinking water is known to cause various cardiovascular diseases in human, exact mechanism still remains elusive. Recently, trivalent-methylated arsenicals, the metabolites of inorganic arsenic, were shown to have higher cytotoxic potential than inorganic arsenic. To study the role of these metabolites in arsenic-induced cardiovascular diseases, we investigated the effect of monomethylarsonous acid (MMA III), a major trivalent-methylated arsenical, on vasomotor tone of blood vessels. In isolated rat thoracic aorta and small mesenteric arteries, MMA III irreversibly suppressed normal vasoconstriction induced by three distinct agonists of phenylephrine (PE), serotonin and endothelin-1. Inhibition of vasoconstriction was retained in aortic rings without endothelium, suggesting that MMA III directly impaired the contractile function of vascular smooth muscle. The effect of MMA III was mediated by inhibition of PE-induced Ca 2+ increase as found in confocal microscopy and fluorimeter in-lined organ chamber technique. The attenuation of Ca 2+ increase was from concomitant inhibition of release from intracellular store and extracellular Ca 2+ influx via L-type Ca 2+ channel, which was blocked by MMA III as shown in voltage-clamp assay in Xenopus oocytes. MMA III did not affect downstream process of Ca 2+, as shown in permeabilized arterial strips. In in vivo rat model, MMA III attenuated PE-induced blood pressure increase indeed, supporting the clinical relevance of these in vitro findings. In conclusion, MMA III-induced smooth muscle dysfunction through disturbance of Ca 2+ regulation, which results in impaired vasoconstriction and aberrant blood pressure change. This study will provide a new insight into the role of trivalent-methylated arsenicals in arsenic-associated cardiovascular diseases.

Tra2β As a Novel Mediator of Vascular Smooth Muscle Diversification

Transformer splicing regulatory proteins determine the sexually dimorphic traits of Drosophila. The role of the vertebrate homologs of Tra-2 in phenotypic specification is undefined. We are using the alternative splicing of the MYPT1 E23 exon as a model for the study of smooth muscle diversification into fast and slow contractile phenotypes. Tra2beta mRNA and protein is expressed at up to 10-fold higher levels in fast smooth muscle tissues such as the rat portal vein and small mesenteric artery, in which E23 is spliced, as compared to the slow smooth muscle tissues of the large arteries and veins, in which E23 is skipped. Tra2beta is upregulated up to 10-fold concordant with the initiation of E23 splicing as the rat portal vein and avian gizzard implement the fast program of gene expression in the perinatal period. In disease models such as portal hypertension and mesenteric artery high/low flow, the portal vein and first order mesenteric artery dynamically downregulate Tra2beta concordant with a shift to E23 skipping and the slow program of gene expression. Tra2beta binds to a highly conserved sequence within E23 and transactivates its splicing in vitro and in vivo; this is abolished with mutation or deletion of this sequence. RNA interference-mediated knockdown of Tra2beta markedly reduces E23 splicing. We propose that Tra2beta has been conserved through evolution and redeployed for the specification of the fast smooth muscle phenotype and may serve as a novel nodal point for the investigation of this process in developmental and disease models.

Phospholipase C-δ1modulates sustained contraction of rat mesenteric small arteries in response to noradrenaline, but not endothelin-1

Vasoconstrictors activate phospholipase C (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP(2)), leading to calcium mobilization, protein kinase C activation, and contraction. Our aim was to investigate whether PLC-delta(1), a PLC isoform implicated in alpha(1)-adrenoreceptor signaling and the pathogenesis of hypertension, is involved in noradrenaline (NA) or endothelin (ET-1)-induced PIP(2) hydrolysis and contraction. Rat mesenteric small arteries were studied. Contractility was measured by pressure myography, phospholipids or inositol phosphates were measured by radiolabeling with (33)Pi or myo-[(3)H]inositol, and caveolae/rafts were prepared by discontinuous sucrose density centrifugation. PLC-delta(1) was localized by immunoblot analysis and neutralized by delivery of PLC-delta(1) antibody. The PLC inhibitor U73122, but not the negative control U-73342, markedly inhibited NA and ET-1 contraction but had no effect on potassium or phorbol ester contraction, implicating PLC activity in receptor-mediated smooth muscle contraction. PLC-delta(1) was present in caveolae/rafts, and NA, but not ET-1, stimulated a rapid twofold increase in PLC-delta(1) levels in these domains. PLC-delta(1) is calcium dependent, and removal of extracellular calcium prevented its association with caveolae/rafts in response to NA, concomitantly reducing NA-induced [(33)P]PIP(2) hydrolysis and [(3)H]inositol phosphate formation but with no effect on ET-1-induced [(33)P]PIP(2) hydrolysis. Neutralization of PLC-delta(1) by PLC-delta(1) antibody prevented its caveolae/raft association and attenuated the sustained contractile response to NA compared with control antibodies. In contrast, ET-1-induced contraction was not affected by PLC-delta(1) antibody. These results indicate the novel and selective role of caveolae/raft localized PLC-delta(1) in NA-induced PIP(2) hydrolysis and sustained contraction in intact vascular tissue.