Mesenteric Vascular Smooth Muscle Cells Research Articles

High blood pressure associates with the remodelling of inward rectifier K+ channels in mice mesenteric vascular smooth muscle cells

The increased vascular tone that defines essential hypertension is associated with depolarization of vascular smooth muscle cells (VSMCs) and involves a change in the expression profile of ion channels promoting arterial contraction. As a major regulator of VSMC resting membrane potential (V(M)), K(+) channel activity is an important determinant of vascular tone and vessel diameter. However, hypertension-associated changes in the expression and/or modulation of K(+) channels are poorly defined, due to their large molecular diversity and their bed-specific pattern of expression. Moreover, the impact of these changes on the integrated vessel function and their contribution to the development of altered vascular tone under physiological conditions need to be confirmed. Hypertensive (BPH) and normotensive (BPN) mice strains obtained by phenotypic selection were used to explore whether changes in the functional expression of VSMC inward rectifier K(+) channels contribute to the more depolarized resting V(M) and the increased vascular reactivity of hypertensive arteries. We determined the expression levels of inward rectifier K(+) channel mRNA in several vascular beds from BPN and BPH animals, and their functional contribution to VSMC excitability and vascular tone in mesenteric arteries. We found a decrease in the expression of Kir2.1, Kir4.1, Kir6.x and SUR2 mRNA in BPH VSMCs, and a decreased functional contribution of both K(IR) and K(ATP) channels in isolated BPH VSMCs. However, only the effect of K(ATP) channel modulators was impaired when exploring vascular tone, suggesting that decreased functional expression of K(ATP) channels may be an important element in the remodelling of VSMCs in essential hypertension.

Abstract 172: Toll-like Receptor-4 Mediates Augmented Contractility In Diabetic Resistance Vessels

Hyperglycemia, the hallmark of diabetes, plays a central role in the pathogenesis of diabetic vascular dysfunction; however the underlying mechanisms are not entirely understood. We previously reported that inhibition of Toll-Like Receptor- 4 (TLR4), a key receptor of the innate immune system, improves vascular function in spontaneously hypertensive rats (SHR) revealing a novel role of TLR4 in the vascular system. In our current study, we hypothesized that upregulation of TLR4 in the vasculature following hyperglycemia leads to augmented vascular contractility. Third order mesenteric arteries from streptozotocin (STZ)-induced diabetic Sprague-Dawley rats (glucose 279 ± 11.6mg/dL) and their respective controls (98 ± 8.03mg/dL ), were incubated with either neutralizing antibody to TLR4 (1ug/mL) or IgG (1ug/mL) for 30 min and contractile responses to phenylephrine (PE) were evaluated ex vivo. Vessels from STZ rats exhibited greater maximal contractile force to PE compared to control (Emax 38.99 ± 1.45 vs. 23.98 ± 0.73 (mN), p<0.05, respectively). Anti-TLR4 incubation attenuated this response to PE in vessels from STZ rats (29.71 ± 1.69mN) but had no effect in control vessels (21.03 ± 1.02mN). To test whether hyperglycemia modulates vascular TLR4, mesenteric vascular smooth muscle cell (MVSM) cultures were stimulated with high glucose (HG 25mM) or normal glucose (NG 5mM) for 48 hours. HG increased protein expression of TLR4 in MVSM compared to MVSM stimulated with NG (2.74±0.34 vs 1.38±0.20, p<0.05). Concurrently, HG caused activation of mitogen-activated protein/extracellular signal-regulated kinase (ERK1/2) (5.04±0.83 vs 3.80±0.90, p<0.05), increased levels of PCNA, a marker of proliferation, (1.34±0.11 vs 0.35±0.04, p<0.05), and decreased levels of calponin, a marker of contractile phenotype, (1.45±0.14 vs 3.62±0.40, p<0.05). Levels of total intracellular reactive oxygen species (ROS) were robustly augmented under HG condition (2.2 fold of the normal glucose). Our results suggest that hyperglycemia upregulates vascular TLR4 leading to augmented vascular contractility, potentially due to activation of ERK1/2, increased ROS and MSMCs proliferation.

CaMKII inhibition in vascular smooth muscle improves angiotensin II–hypertension

The multifunctional calcium/calmodulin‐dependent kinase II (CaMKII) is expressed in vascular smooth muscle cells (VSMC) and activated by Angiotensin‐II (Ang‐II). To test the hypothesis that CaMKII in VSMC mediates Ang‐II‐hypertension, we developed transgenic mice that express the specific CaMKII peptide inhibitor CaMKIIN selectively in VSMC (Tg SM CaMKIIN). These mice have a 65 % reduced CaMKII activity in arteries (p<0.01) and identical baseline blood pressure compared to littermate controls. Following Ang‐II infusion at pressor dose, the increase in mean pressure was significantly attenuated (129±4 mmHg vs, 116±3 mmHg, p<0.05). Ang‐II increases Ca2+ influx through L‐type Ca2+ channels (LTCC). Cytosolic Ca2+ ([Ca2+]) rise leads to an increased vascular tone. Compared with littermates, freshly isolated mesenteric VSMC of Ang‐II treated Tg SM CaMKIIN mice had significantly reduced voltage gated L‐type Ca2+ current (ICa), ICa facilitation. Moreover, the [Ca2+] elevation in response to Ang‐II stimulation was reduced by 50 % in VSMC of TgSM CaMKIIN mice (0.045±0.002 vs, 0.025±0.003; ratio340/380; p<0.05) whereas, level of total releasable SR calcium was decreased by 35 %. In conclusion, inhibition of CaMKII in VSMC significantly attenuates Ang‐II induced hypertension through regulation of LTCC and by restoring the calcium homeostasis. CaMKII may be a novel target in treating hypertension.

Phenylephrine‐induced current and vasoconstriction are blunted in mesenteric arteries of TRPC3 knockout mice

Cationic current mediated by canonical transient receptor potential (TRPC) channels contributes to vascular smooth muscle cell (VSMC) activation. However, the TRPC subtype (TRPC1‐7) that mediates the influx of cations in response to α‐adrenergic receptor activation in the mesenteric circulation has not been established. Here, we used TRPC3 knockout (KO) mice to define the role of TRPC3 channels in phenylephrine (PE) –mediated constriction. Real‐time PCR confirmed the absence of TRPC3 transcript in mesenteric arteries (MA) of KO mice, whereas transcript levels of other TRPC channel subtypes were not significantly different. Diameter reductions in response to KCl (60 mM) were similar between pressurized MA of wild type (WT, 44±5%) and KO (52±9%) mice. However, dose‐dependent contractions to PE were impaired in KO mice (EC50 = 75±4 μM, Emax = 130±7%) compared to WT mice (EC50 = 9±4 μM, Emax = 166±25%). Correspondingly, the mesenteric VSMCs of KO mice showed 52±5% less PE‐evoked current (−1.38±0.16 pA/pF) compared to WT mice (−2.92±0.27 pA/pF). Thus, TRPC3 channels appear to mediate PE‐induced cation entry and vascular tone, consistent with a role in blood pressure regulation. Supported by Grant Z01‐ES101846 from the Intramural Research Program of the NIH (JA), NIH R01 HL064806 (NJR) and NPRP 08‐392‐3‐087 (NJR and KM) from the Qatar National Research Foundation.

Kv1.3 Channels Can Modulate Cell Proliferation During Phenotypic Switch by an Ion-Flux Independent Mechanism

Phenotypic modulation of vascular smooth muscle cells has been associated with a decreased expression of all voltage-dependent potassium channel (Kv)1 channel encoding genes but Kcna3 (which encodes Kv1.3 channels). In fact, upregulation of Kv1.3 currents seems to be important to modulate proliferation of mice femoral vascular smooth muscle cells in culture. This study was designed to explore if these changes in Kv1 expression pattern constituted a landmark of phenotypic modulation across vascular beds and to investigate the mechanisms involved in the proproliferative function of Kv1.3 channels. Changes in Kv1.3 and Kv1.5 channel expression were reproduced in mesenteric and aortic vascular smooth muscle cells, and their correlate with protein expression was electrophysiologicaly confirmed using selective blockers. Heterologous expression of Kv1.3 and Kv1.5 channels in HEK cells has opposite effects on the proliferation rate. The proproliferative effect of Kv1.3 channels was reproduced by "poreless" mutants but disappeared when voltage-dependence of gating was suppressed. These findings suggest that the signaling cascade linking Kv1.3 functional expression to cell proliferation is activated by the voltage-dependent conformational change of the channels without needing ion conduction. Additionally, the conserved upregulation of Kv1.3 on phenotypic modulation in several vascular beds makes this channel a good target to control unwanted vascular remodeling.

Differential Regulation and Recovery of Intracellular Ca2+ in Cerebral and Small Mesenteric Arterial Smooth Muscle Cells of Simulated Microgravity Rat

BackgroundThe differential adaptations of cerebrovasculature and small mesenteric arteries could be one of critical factors in postspaceflight orthostatic intolerance, but the cellular mechanisms remain unknown. We hypothesize that there is a differential regulation of intracellular Ca2+ determined by the alterations in the functions of plasma membrane CaL channels and ryanodine-sensitive Ca2+ releases from sarcoplasmic reticulum (SR) in cerebral and small mesenteric vascular smooth muscle cells (VSMCs) of simulated microgravity rats, respectively.Methodology/Principal FindingsSprague-Dawley rats were subjected to 28-day hindlimb unweighting to simulate microgravity. In addition, tail-suspended rats were submitted to a recovery period of 3 or 7 days after removal of suspension. The function of CaL channels was evaluated by patch clamp and Western blotting. The function of ryanodine-sensitive Ca2+ releases in response to caffeine were assessed by a laser confocal microscope. Our results indicated that simulated microgravity increased the functions of CaL channels and ryanodine-sensitive Ca2+ releases in cerebral VSMCs, whereas, simulated microgravity decreased the functions of CaL channels and ryanodine-sensitive Ca2+ releases in small mesenteric VSMCs. In addition, 3- or 7-day recovery after removal of suspension could restore the functions of CaL channels and ryanodine-sensitive Ca2+ releases to their control levels in cerebral and small mesenteric VSMCs, respectively.ConclusionsThe differential regulation of CaL channels and ryanodine-sensitive Ca2+ releases in cerebral and small mesenteric VSMCs may be responsible for the differential regulation of intracellular Ca2+, which leads to the altered autoregulation of cerebral vasculature and the inability to adequately elevate peripheral vascular resistance in postspaceflight orthostatic intolerance.

Cardiovascular Protection with Danshensu in Spontaneously Hypertensive Rats

The objective of the present study was to evaluate the cardiovascular protective effects of Danshensu, a water-soluble active component of Danshen, in spontaneously hypertensive rats (SHR). SHR (male, 9 weeks old, n=30) were divided into three groups: 1) saline control (n=10); 2) a Danshensu (10 mg/kg/d, intraperitoneally (i.p.)) treatment group (n=10); and 3) a Valsartan (10 mg/kg/d, intragastrically (i.g.)) treatment group (n=10). Age-matched Wistar-Kyoto rats (n=10) were used as normotensive controls. Saline and drug treatments were administered for 6 weeks. When the rats were 15 weeks old, their hearts were excised and arrhythmias were induced by an ex vivo ischemia/reperfusion protocol. The heart weight to body weight index was significantly increased in SHR, and this increase was attenuated with Danshensu treatment (both p<0.05). Systolic blood pressure and diastolic blood pressure were also decreased with Danshensu treatment, from 145±3 and 103±10 mmHg to 116±7 and 87±2 mmHg in SHR and Danshensu-treated groups, respectively (both p<0.05). The incidences of ventricular tachycardia and ventricular fibrillation decreased from 100 to 50% and 30% in SHR, respectively, with Danshensu treatment (both p<0.05). Serum nitric oxide content and inducible nitric oxide synthase activity were significantly increased with Danshensu (both p<0.05). In addition, Danshensu increased the K(+) current density and Ca(2+) activated K(+) channel current density of mesenteric vascular smooth muscle cells isolated from SHRs. Together, these results demonstrate that Danshensu imparts cardiovascular protection by modifying vascular responses during the progression of hypertension.

Mitogen-Activated Protein Kinase–Activated Protein Kinase 2 in Angiotensin II–Induced Inflammation and Hypertension

Vascular oxidative stress and inflammation play an important role in angiotensin II-induced hypertension, and mitogen-activated protein kinases participate in these processes. We questioned whether mitogen-activated protein kinase-activated protein kinase 2 (MK2), a downstream target of p38 mitogen-activated protein kinase, is involved in angiotensin II-induced vascular responses. In vivo experiments were performed in wild-type and Mk2 knockout mice infused intravenously with angiotensin II. Angiotensin II induced a 30 mm Hg increase in mean blood pressure in wild-type that was delayed in Mk2 knockout mice. Angiotensin II increased superoxide production and vascular cell adhesion molecule-1 in blood vessels of wild-type but not in Mk2 knockout mice. Mk2 knockdown by small interfering RNA in mouse mesenteric vascular smooth muscle cells caused a 42% reduction in MK2 protein and blunted the angiotensin II-induced 40% increase of MK2 expression. Mk2 knockdown blunted angiotensin II-induced doubling of intracellular adhesion molecule-1 expression, 2.4-fold increase of nuclear p65, and 1.4-fold increase in Ets-1. Mk2 knockdown abrogated the angiotensin II-induced 4.7-fold and 1.3-fold increase of monocyte chemoattractant protein-1 mRNA and protein. Angiotensin II enhanced reactive oxygen species levels (by 29%) and nicotinamide adenine dinucleotide phosphate oxidase activity (by 48%), both abolished by Mk2 knockdown. Reduction of MK2 blocked angiotensin II-induced p47phox translocation to the membrane, associated with a 53% enhanced catalase expression. Angiotensin II-induced increase of MK2 was prevented by the nicotinamide adenine dinucleotide phosphate oxidase inhibitor Nox2ds-tat. Mk2 small interfering RNA prevented the angiotensin II-induced 30% increase of proliferation. In conclusion, MK2 plays a critical role in angiotensin II signaling, leading to hypertension, oxidative stress via activation of p47phox and inhibition of antioxidants, and vascular inflammation and proliferation.

TRPC6 channels stimulated by angiotensin II are inhibited by TRPC1/C5 channel activity through a Ca2+- and PKC-dependent mechanism in native vascular myocytes

The present work investigated interactions between TRPC1/C5 and TRPC6 cation channel activities evoked by angiotensin II (Ang II) in native rabbit mesenteric artery vascular smooth muscle cells (VSMCs). In low intracellular Ca(2+) buffering conditions (0.1 mm BAPTA), 1 nm and 10 nm Ang II activated both 2 pS TRPC1/C5 channels and 15-45 pS TRPC6 channels in the same outside-out patches. However, increasing Ang II to 100 nm abolished TRPC6 activity but further increased TRPC1/C5 channel activity. Comparison of individual patches revealed an inverse relationship between TRPC1/C5 and TRPC6 channel activity suggesting that TRPC1/C5 inhibits TRPC6 channel activity. Inclusion of anti-TRPC1 and anti-TRPC5 antibodies, raised against intracellular epitopes, in the patch pipette solution blocked TRPC1/C5 channel currents but potentiated by about six-fold TRPC6 channel activity evoked by 1-100 nm Ang II in outside-out patches. Bath application of T1E3, an anti-TRPC1 antibody raised against an extracellular epitope, also increased Ang II-evoked TRPC6 channel activity. With high intracellular Ca(2+) buffering conditions (10 mm BAPTA), 10 nm Ang II-induced TRPC6 channel activity was increased by about five-fold compared to channel activity with low Ca(2+) buffering. In addition, increasing intracellular Ca(2+) levels ([Ca(2+)](i)) at the cytosolic surface inhibited 10 nm Ang II-evoked TRPC6 channel activity in inside-out patches. Moreover, in zero external Ca(2+) (0 [Ca(2+)](o)) 100 nm Ang II induced TRPC6 channel activity in outside-out patches. Pre-treatment with the PKC inhibitor, chelerythrine, markedly increased TRPC6 channel activity evoked by 1-100 nm Ang II and blocked the inhibitory action of [Ca(2+)](i) on TRPC6 channel activity. Co-immunoprecipitation studies shows that Ang II increased phosphorylation of TRPC6 proteins which was inhibited by chelerythrine, 0 [Ca(2+)](o) and the anti-TRPC1 antibody T1E3. These results show that TRPC6 channels evoked by Ang II are inhibited by TRPC1/C5-mediated Ca(2+) influx and stimulation of PKC, which phosphorylates TRPC6 subunits. These conclusions represent a novel interaction between two distinct vasoconstrictor-activated TRPC channels expressed in the same native VSMCs.

Enhanced pressor response to acute Ang II infusion in mice lacking membrane-associated prostaglandin E2 synthase-1

To examine the contribution of vascular membrane-associated prostaglandin E2 synthase-1 (mPGES-1) to acute blood pressure homeostasis. Angiotensin II (AngII, 75 pmol·kg⁻¹·min⁻¹) was continuously infused via the jugular vein into wild-type and mPGES-1(-/-) mice for 30 min, and blood pressure was measured by carotid arterial catheterization. RT-PCR and immunohistochemistry were performed to detect the expression and localization of mPGES-1 in the mouse arterial vessels. Mesenteric arteries were dissected from mice of both genotypes to study vessel tension and measure vascular PGE2 levels. Wild-type and mPGES-1(-/-) mice showed similar blood pressure levels at baseline, and the acute intravenous infusion of AngII caused a greater increase in mean arterial pressure in the mPGES-1(-/-) group, with a similar diuretic and natriuretic response in both groups. mPGES-1 was constitutively expressed in the aortic and mesenteric arteries and vascular smooth muscle cells of wild-type mice. Strong staining was detected in the smooth muscle layer of arterial vessels. Ex vivo treatment of mesenteric arteries with AngII produced more vasodilatory PGE2 in wild-type than in mPGES-1(-/-) mice. In vitro tension assays further revealed that the mesenteric arteries of mPGES-1(-/-) mice exhibited a greater vasopressor response to AngII than those arteries of wild-type mice. Vascular mPGES-1 acts as an important tonic vasodilator, contributing to acute blood pressure regulation.

Role of store-operated calcium entry in imperatorin-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). Imperatorin is known for its potent vasodilatory effects as a dietary furanocoumarin. The study was designed to examine the hypothesis that SOCE have a functional role in imperatorin-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. Imperatorin (1–100 μM) relaxed artery rings 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. Imperatorin didn't inhibit SOCE-mediated increases in cytosolic Ca 2+ levels evoked by the emptying of the stores. In isolated artery rings, imperatorin didn't inhibit SOCE-induced contractions due to store depletion. Our results exclude SOCE mechanism of vasodilatation by imperatorin. But imperatorin is partly similar with nifedipine in vasorelaxation effect.

Raf-1 Kinase Regulates Smooth Muscle Contraction in the Rat Mesenteric Arteries

We investigated the potential role of Raf-1 kinase in mesenteric arterial contraction. Inhibitors of Raf-1 kinase, GW5074, L779450 and ZM 336372 reversed phenylephrine (PE)-induced mesenteric vascular contraction. Studies in vivo in rats showed that GW5074 inhibited PE-induced increase in mean arterial pressure in adult female Sprague-Dawley rats. Isometric tension studies in mesenteric arteries of rats showed that GW5074 did not change the KCl-evoked contraction but significantly inhibited the contractions to PE, 5-HT, U46619, endothelin 1, angiotensin II and phorbol 12, 13-dibutyrate (PDBu). In mesenteric vascular smooth muscle cells (VSMCs), PE stimulated increase in Raf-1 phosphorylation which was inhibited by GW5074. Measurement of [Ca²⁺]_i with Fura-2 showed that GW5074-mediated inhibition of PE-induced contraction was not associated with decreases in [Ca²⁺]_i. VSMCs treated with PE exhibited higher levels of the contractile proteins, p-MYPT1 and p-MLC₂₀, which was inhibited by GW5074. Similarly, PDBu induced increases in phosphorylation of Raf-1, MLC₂₀ and MYPT1 and this was inhibited by GW5074. However, GW5074 did not have any significant effect on PE/PDBu-induced MEK/ERK activation. The results indicate that Raf-1 kinase plays an important role in the regulation of vascular contractility through regulation of calcium sensitization.

Aldosterone-Induced Activation of Signaling Pathways Requires Activity of Angiotensin Type 1a Receptors

Aldosterone has been shown to induce vascular damage, endothelial dysfunction, and myocardial fibrosis, which depend in part on activation of angiotensin II (Ang II)-mediated pathways. However, mechanisms underlying crosstalk between Ang II type 1 receptor (AT(1)R) and mineralocorticoid receptor (MR) are mostly unknown. We tested whether the lack of Ang II type 1a receptor (AT(1a)R) or Ang II type 1b receptor (AT(1b)R) would decrease cellular effects induced by aldosterone. We examined the effect of Ang II or aldosterone after transfection of mesenteric vascular smooth muscle cells (VSMCs) from C57Bl/6 mice with small interference RNA for AT(1a)R, AT(1b)R, or MR for 48 hours. Ang II and aldosterone separately induced ERK1/2, c-Jun NH2-terminal protein kinase (JNK), and nuclear factor (NF)-kappaB phosphorylation after a 20-minute stimulation. Small interference RNA for AT(1a)R downregulated phosphorylation of ERK1/2, JNK, and NF-kappaB after aldosterone stimulation compared to controls. Downregulation of AT(1b)R or MR only abolished the activation of NF-kappaB. In VSMCs from C57Bl/6 mice, aldosterone and Ang II induced the activation of the c-fos promoter from 30 minutes to 1 hour. This effect was blocked when using VSMCs from AT(1a)R knockout mice. We show for the first time that nongenomic and genomic effects of aldosterone are differentially dependent on activity of both AT(1a)R and AT(1b)R. Our data suggest that aldosterone augments AT(1)R-dependent activation of ERK1/2, JNK, and NF-kappaB in VSMCs. We provide mechanistic understanding and experimental in vitro support for the benefit of combination therapy with dual blockade of AT(1)R and MR to treat hypertension and progression of heart failure as reported in clinical studies and animal models.

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.

Vascular α1C and β3 subunits of L‐type calcium channels are coordinately upregulated in the mesenteric arteries of hypertensive C57BL6 mice

Depolarization of vascular smooth muscle cells (VSMCs) opens L‐type calcium (CaL) channels to elicit Ca2+‐dependent contraction. CaL channels consist of pore‐forming α1C and one of four β subunits (β1‐4) that promote α1C expression. β3 is regarded as the major subunit in large arteries, but its functional role in resistance arteries is unclear. We hypothesized that α1C and β3 are coordinately upregulated in hypertension. We infused C57BL6 mice with Angiotensin II (Ang II, 2 ng/g/min, 2 weeks) using osmotic minipumps to establish hypertension. Systolic blood pressures (SBPs) were 170 ± 10 mm Hg in Ang II hypertensive (AHT) mice compared to 114 ± 3 mm Hg in saline infused control mice. Nifedipine (5 mg/kg, i.p.) restored SBP to normal levels in AHT but only mildly lowered SBP in control mice. Isolated mesenteric arteries (MA) from AHT mice showed a 5.1‐fold higher contractile sensitivity to the CaL channel agonist FPL64176 (n=5), and CaL channel current density was increased 1.9‐fold in the mesenteric VSMCs (n=23). Western blots revealed a dual upregulation of α1C and β3 in MA from AHT mice and a marked increase in α1C and β3 immunofluorescence at the membrane of VSMCs. Collectively, our study suggests that: i) CaL channels are primary contributors to hypertension in the AHT mouse, and ii) the α1C/β3 complexes may be the molecular pathways for anomalous Ca2+ influx and vascular tone. Supported by R01 HL064806‐07 (NJR)

Colfibrate attenuates blood pressure and sodium retention in DOCA-salt hypertension

Clofibrate, a peroxisome proliferator-activated receptor-alpha (PPAR alpha) agonist, increases renal tubular cytochrome P450 4a (Cyp4a) expression thereby increasing 20-hydroxyeicosatetraenoic acid (20-HETE) production. To determine if clofibrate affects blood pressure regulation we studied mice with DOCA-salt induced hypertension in wild-type and PPAR alpha knockout mice. Wild-type mice treated with DOCA-salt had higher mean arterial pressures and higher cumulative sodium balance, but lower renal 20-HETE production than did vehicle-treated mice. Treating DOCA-salt mice with clofibrate attenuated the increase in mean arterial pressure and cumulative sodium balance while increasing 20-HETE production and renal Cyp4a expression. In contrast the PPAR alpha knockout mice treated with clofibrate and DOCA-salt showed no attenuation in the increase of blood pressure, cumulative sodium balance, renal 20-HETE production or Cyp4a protein expression. Expression of the PPAR alpha protein was greater in proximal tubules than in renal microvessels. Our results show that PPAR alpha pathway induces renal tubular 20-HETE production which affects sodium retention and blood pressure regulation in DOCA-salt-treated mice.

The vasodilator 17,18‐epoxyeicosatetraenoic acid targets the pore‐forming BK α channel subunit in rodents

17,18-Epoxyeicosatetraenoic acid (17,18-EETeTr) stimulates vascular large-conductance K(+) (BK) channels. BK channels are composed of the pore-forming BK alpha and auxiliary BK beta1 subunits that confer an increased sensitivity for changes in membrane potential and calcium to BK channels. Ryanodine-sensitive calcium-release channels (RyR3) in the sarcoplasmic reticulum (SR) control the process. To elucidate the mechanism of BK channel activation, we performed whole-cell and perforated-patch clamp experiments in freshly isolated cerebral and mesenteric artery vascular smooth muscle cells (VSMC) from Sprague-Dawley rats, BK beta1 gene-deficient (-/-), BK alpha (-/-), RyR3 (-/-) and wild-type mice. The 17,18-EETeTr (100 nm) increased tetraethylammonium (1 mm)-sensitive outward K(+) currents in VSMC from wild-type rats and wild-type mice. The effects were not inhibited by the epoxyeicosatrienoic acid (EET) antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (10 mum). BK channel currents were increased 3.5-fold in VSMC from BK beta1 (-/-) mice, whereas a 2.9-fold stimulation was observed in VSMC from RyR3 (-/-) mice (at membrane voltage 60 mV). The effects were similar compared with those observed in cells from wild-type mice. The BK current increase was neither influenced by strong internal calcium buffering (Ca(2)(+), 100 nm), nor by external calcium influx. The 17,18-EETeTr did not induce outward currents in VSMC BK alpha (-/-) cells. We next tested the vasodilator effects of 17,18-EETeTr on isolated arteries of BK alpha-deficient mice. Vasodilatation was largely inhibited in cerebral and mesenteric arteries isolated from BK alpha (-/-) mice compared with that observed in wild-type and BK beta1 (-/-) arteries. We conclude that 17,18-EETeTr represents an endogenous BK channel agonist and vasodilator. Since 17,18-EETeTr is active in small arteries lacking BK beta1, the data further suggest that BK alpha represents the molecular target for the principal action of 17,18-EETeTr. Finally, the action of 17,18-EETeTr is not mediated by changes of the internal global calcium concentration or local SR calcium release events.

Endothelin-1, but not Ang II, Activates MAP Kinases Through c-Src–Independent Ras-Raf–Dependent Pathways in Vascular Smooth Muscle Cells

Endothelin-1 (ET-1) and angiotensin II (Ang II) activate common signaling pathways to promote changes in vascular reactivity, remodeling, inflammation, and oxidative stress. Here we sought to determine whether upstream regulators of mitogen-activated protein kinases (MAPKs) are differentially regulated by ET-1 and Ang II focusing on the role of c-Src and the small GTPase Ras. Mesenteric vascular smooth muscle cells (VSMCs) from mice with different disruption levels in the c-Src gene (c-Src(+/-) and c-Src(-/-)) and wild-type (c-Src(+/+)) were used. ET-1 and Ang II induced extracellular signal-regulated kinase (ERK) 1/2, SAPK/JNK, and p38MAPK phosphorylation in c-Src(+/+) VSMCs. In VSMCs from c-Src(+/-) and c-Src(-/-), Ang II effects were blunted, whereas c-Src deficiency had no effect in ET-1-induced MAPK activation. Ang II but not ET-1 induced c-Src phosphorylation in c-Src(+/+) VSMCs. Activation of c-Raf, an effector of Ras, was significantly increased by ET-1 and Ang II in c-Src(+/+) VSMCs. Ang II but not ET-1-mediated c-Raf phosphorylation was inhibited by c-Src deficiency. Knockdown of Ras by siRNA inhibited both ET-1 and Ang II-induced MAPK phosphorylation. Our data indicate differential regulation of MAPKs by distinct G protein-coupled receptors. Whereas Ang II has an obligatory need for c-Src, ET-1 mediates its actions through a c-Src-independent Ras-Raf-dependent pathway for MAPK activation. These findings suggest that Ang II and ET-1 can activate similar signaling pathways through unrelated mechanisms. MAP kinases are an important point of convergence for Ang II and ET-1.

Protein phosphatase 2A and Ca 2+-activated K + channels contribute to 11,12-epoxyeicosatrienoic acid analog mediated mesenteric arterial relaxation

Epoxyeicosatrienoic acids (EETs) are considered to be endothelium-derived hyperpolarizing factors, and are potent activators of the large-conductance, Ca 2+-activated K + (BK Ca) channel in vascular smooth muscle. Here, we investigate the signal transduction pathway involved in the activation of BK Ca channels by 11,12-EET and 11,12-EET stable analogs in rat mesenteric vascular smooth muscle cells. 11,12-EET and the 11,12-EET analogs, 11-nonyloxy-undec-8( Z)-enoic acid (11,12-ether-EET-8-ZE), 11-(9-hydroxy-nonyloxy)-undec-8( Z)-enoic acid (11,12-ether-EET-8-ZE-OH) and 11,12- trans-oxidoeicosa-8( Z)-enoic acid (11,12-tetra-EET-8-ZE), caused vasorelaxation of mesenteric resistance arteries. Mesenteric myocyte whole-cell (perforated-patch) currents were substantially (∼150%) increased by 11,12-EET and 11,12-EET analogs. Single-channel recordings were conducted to identify the target for 11,12-EET. 11,12-EET and 11,12-EET analogs also increased mesenteric myocyte BK Ca channel activity in cell-attached patches. Similar results were obtained in cell-free patches. Baseline mesenteric myocyte BK Ca channel activity (NPo) in cell-free patches averaged less than 0.001 at +50 mV and 11,12-EET (1 μmol/L) increased NPo to 0.03 ± 0.02 and 11,12-EET analogs (1 μmol/L) increased NPo to 0.09 ± 0.006. Inhibition of protein phosphatase 2A (PP2A) activity with okadaic acid (10 nmol/L) completely reversed 11,12-EET stimulated BK Ca channel activity and greatly attenuated 11,12-ether-EET-8-ZE mesenteric resistance artery vasorelaxation. 11,12-EET and 11,12-EET analogs increased mesenteric myocyte PP2A activity by 3.5-fold. Okadaic acid and the EET inhibitor, 14,15-epoxyeicosa-5( Z)-enoic acid (14,15-EEZE) inhibited the 11,12-EET mediated increase in PP2A activity. These findings provide initial evidence that PP2A activity contributes to 11,12-EET and 11,12-EET analog activation of mesenteric resistant artery BK Ca channels and vasorelaxation.

Chromosomal Substitution-Dependent Differences in Cardiovascular Responses to Sodium Pentobarbital

In this study we addressed initial laboratory observations of enhanced cardiovascular sensitivity to sodium pentobarbital (PTB) in normotensive Dahl Salt Sensitive rats (SS) compared to Brown Norway (BN) rats. We also used unique consomic (chromosomal substitution) strains to confirm preliminary observations that such differences were related to chromosome 13. Increasing concentrations of PTB were administered sequentially to SS, BN, and SS strains with BN chromosomal substitutions until the point of cardiovascular collapse. Both spontaneous and controlled ventilation were studied. The effect of large (450 microg/mL) and small (35 microg/mL) concentrations of PTB on in situ transmembrane potential of mesenteric arterial vascular smooth muscle (VSM) cells was also measured in these animals with local sympathetic innervation both intact and eliminated. An analysis of variance was used to identify significant differences among groups. Despite virtually identical plasma clearance of PTB, cardiovascular collapse occurred at approximately 35%-45% smaller cumulative doses of administered PTB in SS and other strains compared with BN and SS.13BN (introgression of BN chromosome 13 into an SS) in both spontaneous and controlled ventilation. In neurally intact preparations, large dose PTB-induced VSM hyperpolarization was 4-5 times greater than the small dose in SS and SS.16BN but not in BN and SS.13BN strains. Denervation eliminated this strain difference. These results suggest that enhanced cardiovascular sensitivity to PTB in SS rats is related to greater hyperpolarization of VSM transmembrane potential in resistance vessels and this effect is associated with chromosome 13.