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Mitra Esfandiarei,1 Brikena Hoxha,1 Nicholas A Talley,1 Miranda R Anderson,2 Mustafa F Alkhouli,2 Michaela A Squire,2 Delrae M Eckman,1 Jeganathan Ramesh Babu,3 Gary D Lopaschuk,4 Tom L Broderick21Department of Biomedical Sciences, College of Graduate Studies, Midwestern University, Glendale, AZ, USA; 2Department of Physiology, Laboratory of Diabetes and Exercise Metabolism, College of Graduate Studies, Midwestern University, Glendale, AZ, USA; 3Department of Nutrition, Dietetics, and Hospitality Management, Auburn University, Auburn, AL, USA; 4Cardiovascular Research Centre, Mazankowski Alberta Heart Institute University of Alberta, Edmonton, AB, CanadaBackground: Studies have indicated an association between Alzheimer’s disease (AD) and increased risk of developing cardiovascular complications. Lifestyle modifiable factors, such as exercise and diet, are known to prevent cardio-cerebral disease. Recent studies demonstrate that hearts from early onset triple-transgenic AD mice exhibit pathologies, but it is not clear whether cardiovascular function is altered in this model. Methods: In this study, we measured in vivo cardiovascular function in 7-month-old male 3xTg mice and age-matched wild-type (WT) mice using high-frequency high-resolution ultrasound imaging. Results: Our findings indicated that aortic root measurements and interventricular septal dimensions were similar in 3xTg and wild-type mice. Systolic function, expressed as ejection fraction and fractional shortening, were decreased in 3xTg mice. Late (A) ventricular filling velocities, the early/atrial (E/A) ratio, and mitral valve deceleration time, all indices of diastolic function, were increased in 3xTg mice compared to WT mice. Treadmill exercise training and resveratrol supplementation in the diet for 5 months improved ejection fraction, fractional shortening, and restored diastolic deceleration times. Pulse wave velocity was ∼33% higher in 3xTg, and accompanied by a significant increase in elastin fiber fragmentation within the aortic wall, which was associated with decrease in elastin content and fiber length. Aortic wall and adventitia thickness were increased in 3xTg mice compared to the WT group. Exercise training and resveratrol supplementation, or both, improved overall aortic morphology with no change in pulse wave velocity. Conclusion: Taken together, the results indicate that the aberrations in cardiac function and aortic elastin morphology observed in the 3xTg mouse model of AD can be prevented with exercise training and treatment with resveratrol. The benefits of regular exercise training and resveratrol supplementation of heart and aortic structure in the 3xTg mouse support the value of healthy lifestyle factors on cardiovascular health.Keywords: Alzheimer’s disease, exercise, resveratrol, heart, aorta, ultrasound imaging, pulse wave velocity, aortic wall elasticity

Background: Studies have indicated an association between Alzheimer’s disease (AD) and increased risk of developing cardiovascular complications. Lifestyle modifiable factors, such as exercise and diet, are known to prevent cardio-cerebral disease. Recent studies demonstrate that hearts from early onset triple-transgenic AD mice exhibit pathologies, but it is not clear whether cardiovascular function is altered in this model. Methods: In this study, we measured in vivo cardiovascular function in 7-month-old male 3xTg mice and age-matched wild-type (WT) mice using high-frequency high-resolution ultrasound imaging. Results: Our findings indicated that aortic root measurements and interventricular septal dimensions were similar in 3xTg and wild-type mice. Systolic function, expressed as ejection fraction and fractional shortening, were decreased in 3xTg mice. Late (A) ventricular filling velocities, the early/atrial (E/A) ratio, and mitral valve deceleration time, all indices of diastolic function, were increased in 3xTg mice compared to WT mice. Treadmill exercise training and resveratrol supplementation in the diet for 5 months improved ejection fraction, fractional shortening, and restored diastolic deceleration times. Pulse wave velocity was ~33% higher in 3xTg, and accompanied by a significant increase in elastin fiber fragmentation within the aortic wall, which was associated with decrease in elastin content and fiber length. Aortic wall and adventitia thickness were increased in 3xTg mice compared to the WT group. Exercise training and resveratrol supplementation, or both, improved overall aortic morphology with no change in pulse wave velocity. Conclusion: Taken together, the results indicate that the aberrations in cardiac function and aortic elastin morphology observed in the 3xTg mouse model of AD can be prevented with exercise training and treatment with resveratrol. The benefits of regular exercise training and resveratrol supplementation of heart and aortic structure in the 3xTg mouse support the value of healthy lifestyle factors on cardiovascular health.

Myogenic tone (MT) is a primary modulator of blood flow in the resistance vasculature of the brain, kidney, skeletal muscle, and perhaps in other high-flow organs such as the pregnant uterus. MT is known to be regulated by endothelium-derived factors, including products of the nitric oxide synthase (NOS) and/or the cyclooxygenase (COX) pathways. We asked whether pregnancy influenced MT in myometrial arteries (MA), and if so, whether such an effect could be attributed to alterations in NOS and/or COX. MA (200–300 μm internal diameter, 2–3 mm length) were isolated from 10 nonpregnant and 12 pregnant women undergoing elective hysterectomy or cesarean section, respectively. In the absence of NOS and/or COX inhibition, pregnancy was associated with increased MT in endothelium-intact MA compared with MA from nonpregnant women ( P < 0.01). The increase in MT was not due to increased Ca2+ entry via voltage-dependent channels since both groups of MA exhibited similar levels of constriction when exposed to 50 mM KCl. NOS inhibition ( Nω-nitro-l-arginine methyl ester, l-NAME) or combined NOS/COX inhibition (l-NAME/indomethacin) increased MT in MA from pregnant women ( P = 0.001 and P = 0.042, respectively) but was without effect in arteries from nonpregnant women. Indomethacin alone was without effect on MT in MA from either nonpregnant or pregnant women. We concluded that MT increases in MA during human pregnancy and that this effect was partially opposed by enhanced NOS activity.

Background: The occurrence of AKI in the US continues to grow and causes significant morbidity and mortality. Recent reports indicate stem cell therapy improves renal dysfunction in rodent models of chemically-induced AKI. The intent of this study was to assess whether hAFSCs could be used as an effective therapy for ischemia-induced AKI. Methods: AKI was induced via 1hr bilateral renal ischemia in 3mo, male, nude rats. After ischemia, kidney reperfusion was confirmed for 20min followed by injection of either saline (saline, 0.05ml) or hAFSC (cell, 1.25x106 cells/pole) into the upper and lower pole of both kidneys. The surgical incision was closed immediately after saline/cell injection and rats were closely monitored during recovery. Serum creatinine (sCR) was measured 24hr prior to injection (B) and for 7 days post cell/saline injection (d1-d7). The effect of cell therapy on renal and glomerular structure/integrity, SOD and Caspase-3 expression was evaluated histologically. Serum and histological assess...

P2Y12 is the major platelet receptor that mediates ADP-induced aggregation. P2Y12 is also expressed by vascular cells. The factors that regulate P2Y12 expression have not been determined. Since nicotine (NIC) has effects on platelet activation and vascular function, and because nicotinic and purinerigic receptors may interact, we determined whether nicotine altered P2Y12 expression. Four cell lines (human coronary artery endothelial cells, HCAEC; human umbilical vein endothelial cells, HUVEC; human aortic smooth muscle cells, HASMC; and human megakaryoblastic cells, MEG-01) were cultured in the absence or presence of nicotine. Immunoblotting for P2Y12, P2Y2, and actin was performed. Nicotine, at concentrations of 0.1–1.0 μM, induced P2Y12 (but not P2Y2) expression in all the four cell lines. HASMC exhibited the greatest induction with a sixfold mean increase in P2Y12 expression in response to 0.25 μM nicotine. The induction was inhibited by nicotinic acetylcholine receptor antagonists. Healthy smokers were observed to have higher P2Y12 expression in platelet lysates compared to non-smokers. Nicotine induces the expression of P2Y12 in vascular cells and megakaryoblasts, and is mediated by nicotinic acetylcholine receptors. Smokers exhibit higher platelet P2Y12, possibly mediated via nicotine. These results may contribute to a better understanding of the effects of cigarette smoking on platelet activation and the vessel wall. Condensed Abstract The factors that regulate the expression of P2Y12, the platelet ADP receptor, have not been determined. Four cell lines (human coronary artery endothelial cells, HCAEC; human umbilical vein endothelial cells, HUVEC; human aortic smooth muscle cells, HASMC; and human megakaryoblastic cells, MEG-01) were cultured in the absence or presence of nicotine. Nicotine, at concentrations of 0.1–1.0 μM, induced P2Y12 expression in all the four cell lines. HASMC exhibited the greatest induction with a sixfold mean increase in P2Y12 expression in response to 0.25 μM nicotine. The induction was inhibited by nicotinic acetylcholine receptor antagonists. Healthy smokers were observed to have higher P2Y12 expression in platelet lysates compared to non-smokers. These results may contribute to a better understanding of the effects of cigarette smoking on platelet activation and the vessel wall.

Controversy exists concerning whether activation of N -methyl- d -aspartate (NMDA) receptors exerts direct dilator effects on cerebral arteries. The purpose of this study was to examine the responses of isolated piglet arteries to NMDA to determine whether isolated arteries, apart from surrounding neuronal tissue, are capable of responding to NMDA. Piglet arteries (100–200 μm) were isolated from branches of the middle cerebral artery and carefully dissected free of adherent tissue. Arteries were then mounted in an arteriograph system and pressurized to either 30 mm Hg ( n = 8), 60 mm Hg ( n = 10), 80 mm Hg ( n = 6), or 100 mm Hg ( n = 5). After development of spontaneous tone, NMDA (10 −5 to 10 −3 M) was administered abluminally to the vessels, and no appreciable response was noted (for example; 10 −4 M, 30 mm Hg: 3 ± 3% change in active diameter; 60 mm Hg: −4 ± 3% change in active diameter). Following a thorough washout, vessels were treated with bradykinin (10 −9 to 10 −7 M), and the arteries did respond (10 −7 M, 30 mm Hg: 26 ± 3% change in active diameter; 60 mm Hg: 65 ± 10% change in active diameter). In contrast, 10 −5 M and 10 −4 M NMDA dilated arteries in vivo by 9 ± 2% and 29 ± 6% change in active diameter, respectively ( n = 6). These results demonstrate that isolated cerebral arteries do not respond directly to NMDA receptor activation. This work confirms our previous in vivo data and is consistent with the hypothesis that cerebral arteries respond to NMDA through a secondary interaction mediated by neuronal release of NO and not to NMDA directly.

Spontaneous transient outward K+ currents (STOCs) elicited by Ca2+ sparks and steady-state K+ currents modulate vascular reactivity, but effects of artery size, diabetic dyslipidemia, and exercise on these differentially regulated K+ currents are unclear. We studied the conduit arteries and microvessels of male Yucatan swine assigned to one of three groups for 20 wk: control (C, n = 7), diabetic dyslipidemic (DD, n = 6), or treadmill-trained DD animals (DDX, n = 7). Circumflex artery blood flow velocity obtained with intracoronary Doppler and lumen diameters obtained by intravascular ultrasound enabled calculation of absolute coronary blood flow (CBF). Ca2+ sparks were determined in pressurized microvessels, and perforated patch clamp assessed K+ current in smooth muscle cells isolated from conduits and microvessels. Baseline CBF in DD was decreased versus C. In pressurized microvessels, Ca2+ spark activity was significantly lower in DD versus C and DDX ( P < 0.05 vs. DDX). STOCs were pronounced in microvessel (∼35 STOCs/min) in sharp contrast to conduit cells (∼2 STOCs/min). STOCs were decreased by 86% in DD versus C and DDX in microvessels; in contrast, there was no difference in STOCs across groups in conduit cells. Steady-state K+ current in microvessels was decreased in DD and DDX versus C; in contrast, steady-state K+ current in conduit cells was decreased in DDX versus DD and C. We conclude that steady-state K+ current and STOCs are differentially regulated in conduit versus microvessels in health and diabetic dyslipidemia. Exercise prevented diabetic dyslipidemia-induced decreases in baseline CBF, possibly via STOC-regulated basal microvascular tone.

Cysteinyl leukotrienes activate the cysteinyl leukotriene type 1 receptor (CysLT1R) to regulate numerous cell functions important in inflammatory processes and diseases such as asthma. Despite its physiologic importance, no studies to date have examined the regulation of CysLT1R signaling or trafficking. We have established model systems for analyzing recombinant human CysLT1R and found regulation of internalization and signaling of the CysLT1R to be unique among G protein-coupled receptors. Rapid and profound LTD4-stimulated internalization was observed for the wild type (WT) CysLT1R, whereas a C-terminal truncation mutant exhibited impaired internalization yet signaled robustly, suggesting a region within amino acids 310-321 as critical to internalization. Although overexpression of WT arrestins significantly increased WT CysLT1R internalization, expression of dominant-negative arrestins had minimal effects, and WT CysLT1R internalized in murine embryonic fibroblasts lacking both arrestin-2 and arrestin-3, suggesting that arrestins are not the primary physiologic regulators of CysLT1Rs. Instead, pharmacologic inhibition of protein kinase C (PKC) was shown to profoundly inhibit CysLT1R internalization while greatly increasing both phosphoinositide (PI) production and calcium mobilization stimulated by LTD4 yet had almost no effect on H1 histamine receptor internalization or signaling. Moreover, mutation of putative PKC phosphorylation sites within the CysLT1R C-tail (CysLT1RS(313-316)A) reduced receptor internalization, increased PI production and calcium mobilization by LTD4, and significantly attenuated the effects of PKC inhibition. These findings characterized the CysLT1R as the first G protein-coupled receptor identified to date in which PKC is the principal regulator of both rapid agonist-dependent internalization and rapid agonist-dependent desensitization.

Novel non-thiazolidinedione, tyrosine-derived peroxisome proliferator-activated receptor γ agonists, GI 262570, GW 7845, GW 1929, developed by GlaxoSmithKline (GSK) along with pioglitazone and nisoldipine, were studied on currents through L-type voltage-dependent calcium channels (VDCC) in freshly isolated smooth muscle cells from mesenteric arteries, and on the diameter of pressurized mesenteric arteries in vitro. Using Ba2+ (10 mmol/l) as the charge carrier through VDCC, the half-inhibition constants (IC50) for GI 262570, GW 7845, GW 1929, and pioglitazone were 2.0 ± 0.5, 3.0 ± 0.5, 5.0 ± 0.7, and 10.0 ± 0.8 µmol/l, respectively. For arterial diameter measurements the IC50 values for GI 262570, GW 7845, GW 1929, and pioglitazone were 2.4, 4.1, 6.3, and 13.9 µmol/l, respectively. Each GSK compound and pioglitazone was effective at inhibiting VDCC and relaxing pressurized arteries, suggesting that the vasodilation of resistance arteries could be explained by the inhibition of calcium entry through VDCC.

20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P-450 metabolite of arachidonic acid, is a vasoconstrictor in the systemic circulation and a vasodilator in the adult pulmonary circulation. Little is known about the vasoactive properties of 20-HETE in the newborn pulmonary circulation. The objectives of this study were to determine the vascular effects of 20-HETE and to explore the signaling mechanism(s) that mediate these effects in newborn pulmonary resistance-level arteries (PRA). Our findings demonstrate that, in contrast to the adult pulmonary circulation where 20-HETE mediates vasodilation, it causes constriction in newborn PRA at resting tone. Furthermore, inhibition of cyclooxygenase (COX) with indomethacin augments 20-HETE-induced constriction. The enhanced constrictor response to 20-HETE under conditions of COX inhibition is abolished in endothelium-disrupted PRA, suggesting that 20-HETE either stimulates endothelium-derived COX to release a counteracting vasodilator or is rapidly metabolized by COX to a less potent vasoconstrictor. 20-HETE-induced constriction is significantly inhibited by blocking calcium-dependent K+(KCa) channels and the thromboxane-PGH2receptor. Altogether, our data indicate that the vascular actions of 20-HETE are partially mediated via the activation of KCachannels and are significantly modulated by interactions with the COX-prostaglandin pathway.

The transcription factor NFAT (nuclear factor of activated T-cells) is implicated in cardiac hypertrophy and vasculogenesis. NFAT activation, reflecting dephosphorylation by the calcium-dependent phosphatase, calcineurin, and subsequent nuclear localization, is generally thought to require a sustained increase in intracellular calcium. However, in smooth muscle we have found that elevation of calcium by membrane depolarization fails to induce an increase in nuclear localization of the NFATc3 isoform. Here, we demonstrate that physiological intravascular pressure (100 mm Hg) induces an increase in NFATc3 nuclear localization in mouse cerebral arteries. Pressure-induced NFATc3 nuclear accumulation is abrogated by endothelial denudation and by nitric-oxide synthase, cGMP-dependent kinase (PKG), and voltage-dependent calcium channels inhibition. We further show that exogenous nitric oxide, in combination with an elevation in calcium, is an effective stimulus for NFATc3 nuclear accumulation. c-Jun terminal kinase 2 (JNK) activity, which has been shown to regulate NFATc3 nuclear export, is also reduced by pressure, an effect that is prevented by pretreatment with a PKG inhibitor. Consistent with this, pressure-induced NFATc3 nuclear accumulation is independent of PKG in arteries from JNK2(-/-) mice. Collectively, our results indicate that both activation of the NO/PKG pathway and elevation of smooth muscle calcium are required for NFATc3 nuclear accumulation and that PKG inhibits JNK2 to decrease NFAT nuclear export. Our findings suggest that at physiological intravascular pressures NFATc3 is localized to the nucleus in smooth muscle cells of intact arteries and indicate a novel and unexpected role for nitric oxide/PKG in NFAT activation.

Locally injected skeletal muscle precursor cells (skMPCs) integrate into and restore the muscle layers, innervation, vasculature, and function of the sphincter complex in animal models of intrinsic urinary sphincter deficiency (ISD). The goal of the present study was to test the dose-effect safety profile of skMPC therapy in a dog model of ISD. Sphincter deficiency was created in 20 adult female dogs by surgically removing the skeletal muscle layer of the urinary sphincter complex. skMPCs isolated from the hind leg were expanded in culture and injected 4 weeks later into the sphincter complex at a dose of 25 million cells (n = 5), 50 million cells (n = 5), or 100 million cells (n = 5) per milliliter in a 2-ml volume. Five dogs received no sphincter injection. The measures of maximal sphincter pressure, complete blood count, and blood chemistry were performed monthly until their sacrifice at 9 months. At that point, full necropsy was performed to assess the safety of the skMPC injections. Injection of different doses of cells had no effects on the body weight, blood cell count, or kidney or liver function test results (p > .05 among the skMPC doses). Some incidental pathologic features were found in the lower urinary tract in all groups and were most likely associated with repeat catheterization. The maximal urinary sphincter pressure was higher in the 50 million cells per milliliter treatment group than in the other experimental groups (p < .05). The findings of the present study have confirmed that urinary sphincter injection of skMPCs results in no significant local or systemic pathologic features within the dose range that improves sphincter pressures.

X-linked myotubular myopathy (XLMTM) is an isogenic muscle disease characterized by progressive wasting of skeletal muscle, weakness, and premature death of affected male offspring. Recently, the XLMTM gene knock-in mouse, Mtm1 p.R69C, was found to have a similar phenotype as the MTM1 gene mutation in humans (e.g., central nucleation of small myofibers, attenuated muscle strength, and motor unit potentials). Using this rodent model, we investigated whether syngeneic cell therapy could mitigate muscle weakness. Donor skeletal muscle-derived myoblasts were isolated from C57BL6 wild-type (WT) and Mtm1 p.R69C (KI) mice for transplantation into the gastrocnemius muscle of recipient KI mice. Initial experiments demonstrated that donor skeletal muscle-derived myoblasts from WT and KI mice remained in the gastrocnemius muscle of the recipient KI mouse for up to 4 weeks posttransplantation. KI mice receiving syngeneic skeletal muscle-derived myoblasts displayed an increase in skeletal muscle mass, augmented force generation, and increased nerve-evoked skeletal muscle action potential amplitude. Taken together, these results support our hypothesis that syngeneic cell therapy may potentially be used to ameliorate muscle weakness and delay the progression of XLMTM, as application expands to other muscles.

Small arteries exhibit tone, a partially contracted state that is an important determinant of blood pressure. In arterial smooth muscle cells, intracellular calcium paradoxically controls both contraction and relaxation. The mechanisms by which calcium can differentially regulate diverse physiological responses within a single cell remain unresolved. Calcium-dependent relaxation is mediated by local calcium release from the sarcoplasmic reticulum. These 'calcium sparks' activate calcium-dependent potassium (BK) channels comprised of alpha and beta1 subunits. Here we show that targeted deletion of the gene for the beta1 subunit leads to a decrease in the calcium sensitivity of BK channels, a reduction in functional coupling of calcium sparks to BK channel activation, and increases in arterial tone and blood pressure. The beta1 subunit of the BK channel, by tuning the channel's calcium sensitivity, is a key molecular component in translating calcium signals to the central physiological function of vasoregulation.

Acetylcholine (ACh) elicits an endothelium-dependent relaxation and hyperpolarization in the absence of nitric oxide (NO) and prostaglandin synthesis in the guinea-pig coronary artery (GPCA). This response has been attributed to a factor termed endothelial-derived hyperpolarizing factor (EDHF). Recently it has been suggested that EDHF may be a cytochrome P450 product of arachidonic acid (AA) i.e., an epoxyeicosatrienoic acid (EET). The present study investigated whether this pathway could account for the response to ACh observed in the GPCA in the presence of 100 μM Nω-nitro-L-arginine and 10 μM indomethacin. ACh, AA and 11,12-EET each produced concentration-dependent relaxations in arteries contracted with the H1-receptor agonist AEP (2,2-aminoethylpyridine). The AA-induced relaxation was significantly enhanced in the presence of the cyclo-oxygenase/lipoxygenase inhibitor, eicosatetranynoic acid (30 μM). The cytochrome P450 inhibitors proadifen (10 μM) and clotrimazole (10 μM) inhibited ACh, lemakalim (LEM) and AA-induced relaxation, whereas 17-octadecynoic acid (100 μM) and 7-ethoxyresorufin (10 μM) were without effect on all three vasodilators. Proadifen and clotrimazole also inhibited ACh (1 μM) and LEM (1 μM)-induced hyperpolarization. The ability of various potassium channel blockers to inhibit relaxation responses elicited with ACh, AA and 11,12-EET was also determined. Iberiotoxin (IBTX; 100 nM) was without effect on responses to ACh but significantly reduced responses to both AA and 11,12-EET. In contrast, 4-aminopyridine (4-AP; 5 mM) significantly reduced response to ACh but not responses to AA and 11,12-EET. Combined IBTX plus (4-AP) inhibited the ACh-induced relaxation to a greater extent than 4-AP alone. Apamin (1 μM), glibenclamide (10 μM) and BaCl2 (50 μM) had no significant effect on responses to ACh, AA and 11,12-EET. IBTX (100 nM) significantly reduced both 11,12-EET (33 μM) and AA (30 μM) hyperpolarization without affecting the ACh (1 μM)-induced hyperpolarization. In contrast, 4-AP significantly reduced the ACh-induced hyperpolarization without affecting either AA or 11,12-EET-induced hyperpolarizations. In summary, our results suggest that the coronary endothelium releases a factor upon application of AA which hyperpolarizes the smooth muscle. The similarity of pharmacology between AA and 11,12-EET suggests that this factor is an EET. However, the disparity of pharmacology between responses to ACh versus responses to 11,12-EET do not support the hypothesis that EETs represent the predominant factor which ACh releases from the endothelium that leads to NO- and prostaglandin-independent hyperpolarization and relaxation in the GPCA.

Background Doxorubicin (DOX) is associated with premature cardiovascular events including myocardial infarction. This study was performed to determine if the weekly administration of DOX influenced coronary arteriolar medial and/or adventitial wall thickening. Methods Thirty-two male Sprague-Dawley rats aged 25.1± 2.4 weeks were randomly divided into three groups and received weekly intraperitoneal injections of normal saline (saline, n = 7), or low (1.5 mg/kg to 1.75 mg/kg, n = 14) or high (2.5 mg/kg, n = 11) doses of DOX. The animals were treated for 2–12 weeks, and euthanized at pre-specified intervals (2, 4, 7, or 10+ weeks) to obtain histopathologic assessments of coronary arteriolar lumen diameter, medial wall thickness, adventitial wall thickness, and total wall thickness (medial thickness + adventitial thickness). Results Lumen diameter was similar across all groups (saline: 315±34 µm, low DOX: 286±24 µm, high DOX: 242±27 µm; p = 0.22). In comparison to animals receiving weekly saline, animals receiving weekly injections of 2.5 mg/kg of DOX experienced an increase in medial (23±2µm vs. 13±3µm; p = 0.005), and total wall thickness (51±4µm vs. 36±5µm; p = 0.022), respectively. These increases, as well as adventitial thickening became more prominent after normalizing for lumen diameter (p

Purpose Botulinum neurotoxin-A (BoNTA) is used to treat several disorders, including Raynaud phenomenon. Recent investigations cite toxin-induced increases in blood flow, but no mechanism for BoNTA's actions is proposed. This study hypothesized that local application of BoNTA causes arteriolar vasodilation through sympathetic blockade and results in increased blood flow. Methods Microvascular effects of BoNTA were assessed using a rat cremaster preparation. Cremaster microvascular diameters were measured in the muscle before and after treatment with the muscle paralytic agent gallamine triethiodide. Preparations were then treated with one of the following: BoNTA (4, 6, or 10 units), BoNTA dilution vehicle, or denatured BoNTA. Arteriolar diameters were measured repeatedly over the observation period. Additional preparations were treated with either tetrodotoxin or prazosin and rauwolscine before BoNTA to confirm that the observed vasodilatory responses were the result of sympathetic neural inhibition. Results The BoNTA application resulted in a significant dose-dependent vasodilation (13% to 15%) of observed cremaster arterioles. Control treatments did not cause vasodilation. Both tetrodotoxin and prazosin/rauwolscine treatments elicited similar vasodilatory effects, with no additional vasodilation elicited by BoNTA. Addition of sodium nitroprusside following BoNTA elicited further vasodilation. In addition, systemic arterial pressure was unaffected by the local administration of BoNTA. Conclusions Local application of BoNTA results in arteriolar dilation that yields an approximate 69% increase in blood flow, without changing systemic arterial pressure. A BoNTA-mediated vasodilation through sympathetic blockade is a likely mechanism to explain the increase in blood flow reported after treatment with the toxin. Clinical relevance The ability of BoNTA to inhibit sympathetic nervous input reduces vasoconstriction, which is the most likely mechanism for improvement seen in Raynaud phenomenon patients following BoNTA injection.

Although the use of antenatal glucocorticoids has resulted in decreased neonatal morbidity/mortality, recent animal studies have raised concerns regarding adverse effects of these medications on postnatal cardiovascular function. We hypothesized that antenatal betamethasone (Beta) exposure alters cerebral vascular reactivity in adult female sheep. We observed that K+-induced constriction was comparable in middle cerebral artery (MCA) from Beta-exposed animals and age-matched controls. Pressure-induced constriction was significantly attenuated in MCA from Beta-exposed compared with control sheep. Inhibition of NOS significantly augmented pressure-induced constriction in MCA from both Beta-exposed and control sheep whereas cyclooxygenase (COX) inhibition augmented pressure-induced constriction only in MCA from Beta-exposed sheep. Furthermore, NOS and COX inhibition significantly attenuated bradykinin (BK)-induced dilation in MCA from both Beta-exposed and control sheep. However, there appeared to be a greater contribution of both NOS and COX to BK-induced dilation in Beta-exposed compared with control MCA. Our findings demonstrate that fetal exposure to a clinically relevant course of Beta alters cerebral vascular tone and reactivity in adult female sheep.

Niacin is suboptimally used in patients because it causes flushing and erythema. These side effects have been attributed to release of the vasodilating prostaglandin D2, generated in a reaction catalyzed by cyclooxygenase-1. Aspirin reduces but does not completely eliminate these side effects. Because some patients are resistant to its antiplatelet effects, we hypothesized that patients with persistent niacin-induced erythema might be aspirin resistant.Platelet function studies (via the use of a whole-blood platelet function aggregometer [VerifyNow; Accumetrics, San Diego, CA] with end points of aspirin reaction unit [ARUs] and P2Y12 reaction units [PRUs]) were performed on 32 healthy, drug-free subjects before and after 324 mg of aspirin. A niacin skin test with the use of topical methylnicotinate was also performed before and after the administration of aspirin. Responses to methyl nicotinate were assessed by a reaction score and by counting the time to first visible redness (TTR).All subjects had an expected decrease in arachidonic acid induced platelet response (ARU 642.8 ± 47.20 before to 431.5 ± 41.1 after aspirin, P.0001) without a significant change in the PRU. The reaction score and TTR were prolonged by aspirin at methylnicotinate concentrations ≥ 0.001 M. Although no subject had aspirin resistance (defined as ARU550), there was considerable variability in skin responses with erythema elicited in all subjects at the greatest concentrations. There was no difference in the ARU for subjects with TTR values above and below the mean, indicating that aspirin resistance does not explain the variation in skin responses to a topical niacin derivative.Aspirin resistance is unlikely to be a significant contributor to the persistent erythema and flushing in niacin-treated patients.

Leukotrienes are important lipid mediators of asthma that contribute to airway inflammation and bronchoconstriction. Critical mechanisms for physiological regulation of the main G protein-coupled receptor (GPCR) mediating the leukotriene responses in asthma, cysteinyl leukotriene type 1 receptor (CysLT1R), have not been delineated. Although desensitization of GPCRs is a well-established phenomenon, studies demonstrating its physiological relevance are lacking. Here, we demonstrate that relief of PKC-mediated desensitization of endogenous CysLT1Rs augments multiple LTD4-stimulated cellular functions, with associated increases in intracellular signaling events. In analyses of airway smooth muscle contraction ex vivo, PKC inhibition augmented LTD4-stimulated contraction, and increased phosphoinositide hydrolysis and calcium flux in both murine and human airway smooth muscle cells. Similarly, for human monocytes, PKC inhibition augmented LTD4-stimulated calcium flux and cell migration assessed in transwell chamber experiments and also augmented LTD4-induced production of monocyte chemotactic protein assessed by ELISA. In contrast, PKC inhibition had no effect or slightly attenuated these cell functions and signaling events promoted by other receptor agonists, suggesting that despite antithetical effects on downstream events, desensitization of the CysLT1R is the principal mechanism by which PKC regulates the functional consequences of CysLT1R activation.

The transcription factor NFAT (nuclear factor of activated T-cells) is implicated in cardiac hypertrophy and vasculogenesis. NFAT activation, reflecting dephosphorylation by the calcium-dependent phosphatase, calcineurin, and subsequent nuclear localization, is generally thought to require a sustained increase in intracellular calcium. However, in smooth muscle we have found that elevation of calcium by membrane depolarization fails to induce an increase in nuclear localization of the NFATc3 isoform. Here, we demonstrate that physiological intravascular pressure (100 mm Hg) induces an increase in NFATc3 nuclear localization in mouse cerebral arteries. Pressure-induced NFATc3 nuclear accumulation is abrogated by endothelial denudation and by nitric-oxide synthase, cGMP-dependent kinase (PKG), and voltage-dependent calcium channels inhibition. We further show that exogenous nitric oxide, in combination with an elevation in calcium, is an effective stimulus for NFATc3 nuclear accumulation. c-Jun terminal kinase 2 (JNK) activity, which has been shown to regulate NFATc3 nuclear export, is also reduced by pressure, an effect that is prevented by pretreatment with a PKG inhibitor. Consistent with this, pressure-induced NFATc3 nuclear accumulation is independent of PKG in arteries from JNK2(-/-) mice. Collectively, our results indicate that both activation of the NO/PKG pathway and elevation of smooth muscle calcium are required for NFATc3 nuclear accumulation and that PKG inhibits JNK2 to decrease NFAT nuclear export. Our findings suggest that at physiological intravascular pressures NFATc3 is localized to the nucleus in smooth muscle cells of intact arteries and indicate a novel and unexpected role for nitric oxide/PKG in NFAT activation.

Abstract —The molecular bases of inwardly rectifying K + (Kir) currents and K + -induced dilations were examined in cerebral arteries of mice that lack the Kir2.1 and Kir2.2 genes. The complete absence of the open reading frame in animals homozygous for the targeted allele was confirmed. Kir2.1 −/− animals die 8 to 12 hours after birth, apparently due to a complete cleft of the secondary palate. In contrast, Kir2.2 −/− animals are viable and fertile. Kir currents were observed in cerebral artery myocytes isolated from control neonatal animals but were absent in myocytes from Kir2.1 −/− animals. Voltage-dependent K + currents were similar in cells from neonatal control and Kir2.1 −/− animals. An increase in the extracellular K + concentration from 6 to 15 mmol/L caused Ba 2+ -sensitive dilations in pressurized cerebral arteries from control and Kir2.2 mice. In contrast, arteries from Kir2.1 −/− animals did not dilate when the extracellular K + concentration was increased to 15 mmol/L. In summary, Kir2.1 gene expression in arterial smooth muscle is required for Kir currents and K + -induced dilations in cerebral arteries. (Circ Res. 2000;87:160-166.)

1. The effects of acetylcholine (ACh) on membrane potential, relaxation and cyclic GMP levels were compared to the NO donor L-nitrosocysteine (Cys-NO) in segments of guinea-pig coronary artery. 2. ACh and Cys-NO produced concentration-dependent relaxations of muscles contracted with the H1 receptor agonist, 2-(2-aminoethyl)pyridine (AEP, 0.35 mM). The relaxation to ACh was unchanged in the presence of NG-monomethyl-L-arginine (L-NMMA; 350 microM) or indomethacin (3 microM). 3. Oxyhaemoglobin (HbO; 20 microM) alone or in combination with L-NMMA increased the EC50 for ACh-induced relaxation whereas relaxation with Cys-NO was almost completely abolished with HbO. 4. Scorpion venom (SV; 8.7 micrograms ml-1) increased the EC50 for relaxation with ACh but not Cys-NO. Combined L-NMMA, HbO and SV produced nearly complete abolition of ACh-induced relaxations. 5. Basal cyclic GMP levels (i.e., 20 pmol mg-1 protein) were significantly increased following addition of either ACh (190 pmol mg-1 protein) or Cys-NO (240 pmol mg-1 protein). L-NMMA significantly reduced the rise of cyclic GMP with ACh but not Cys-NO. In contrast, SV did not significantly reduce the rise in cyclic GMP produced with ACh. In the combined presence of L-NMMA and HbO neither ACh nor Cys-NO produced a significant increase in cyclic GMP levels. 6. ACh gave rise to significantly greater membrane hyperpolarization than Cys-NO both in the presence and absence of AEP. Combined L-NMMA and HbO did not reduce the amplitude of hyperpolarization with ACh. 7. These data indicate that some but not all of the actions of ACh in the coronary artery can be mimicked by the NO donor, Cys-NO, suggesting that ACh releases NO as well as a second hyperpolarizing factor (i.e., EDHF). Release of NO results in a large increase in tissue cyclic GMP levels and minimal change in membrane potential whereas release of EDHF results in a large membrane hyperpolarization which is independent of changes in tissue cyclic GMP levels. Both of these pathways appear to contribute to relaxation throughout the entire ACh concentration-relaxation relationship.

Analogues of L-arginine with modifications at the terminal guanidino nitrogen and/or the carboxyl terminus of the molecule have been widely used for their ability to inhibit the production of nitric oxide and are thought to be competitive antagonists of nitric oxide synthase. The present studies were designed to test the possibility that these agents are also muscarinic receptor antagonists. Acetylcholine produced concentration-dependent contraction of endothelium-denuded rabbit coronary artery as well as isolated strips of canine colonic smooth muscle. The arginine analogue NG-nitro L-arginine methyl ester (L-NAME, 100 microM) but not NG-monomethyl L-arginine (L-NMMA, 100 microM) significantly shifted these contractile relations to the right, an effect that was not reversed by addition of 1 mM L-arginine. In radioligand binding studies using the muscarinic radioligand [3H]quinuclidinyl benzilate and tissues known to contain differing contributions of M1, M2, and M3 muscarinic receptors, addition of increasing concentrations of L-NAME resulted in a monophasic competition of binding with affinities (Ki) ranging from 68 microM in endothelium to 317 microM in whole aorta. Addition of the hydrolysis-resistant guanosine 5'-triphosphate analogue GTP gamma S (100 microM) had no effect on L-NAME competition of [3H]quinuclidinyl benzilate binding. Addition of L-NAME in radioligand binding competition studies using the agonist carbachol did not result in an alteration of the receptor's affinity for agonist, confirming the competitive nature of the interaction of L-NAME with the muscarinic receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

External potassium ions have long been known as mediators of vasodilation of several vascular beds, including the coronary and cerebral circulations.1 2 3 4 5 6 Indeed, potassium ions have been viewed as communicators of the metabolic state of the cells that surround blood vessels. For example, release of potassium ions from neurons is communicated through glial cells to regulate cerebral artery diameter.7 Recently, it has been suggested that the potassium ions from endothelial cells may signal smooth muscle to relax and, as such, may constitute an endothelial-derived hyperpolarizing factor.8 Two targets of external potassium ions have been proposed: the Na+/K+ ATPase and the inward rectifier potassium channel.1 5 9 An elevation of external potassium causes very different responses of these two molecular targets. The electrogenic Na+/K+ ATPase is activated by external potassium with a half-activation constant of about 1 to 2 …

1. The contractile and electrical responses to acetylcholine (ACh) in isolated segments of guinea-pig and rabbit coronary arteries were compared to those of the putative adenosine 5'-triphosphate (ATP)-dependent K+ channel opener, BRL 38227. 2. Both ACh and BRL 38227 produced concentration-dependent relaxation of vessel segments contracted with the H1-receptor agonist, 2-(2-aminoethyl)pyridine. 3. An IC90 of either vasodilator also produced 17-20 mV of hyperpolarization of the guinea-pig coronary artery. 4. Glibenclamide (1-35 microM) depolarized the guinea-pig coronary artery by 8-12 mV and antagonized BRL 38227- but not ACh-induced relaxation and hyperpolarization. 5. In the guinea-pig coronary artery, the K+ channel blockers phencyclidine (PCP, 100 microM), tetraethylammonium (TEA, 10 mM) and scorpion venom (8.7 micrograms ml-1) all significantly reduced ACh-induced relaxation and hyperpolarization whereas only PCP was an effective antagonist of both relaxation and hyperpolarization with BRL 38227. 6. Similar effects of glibenclamide and scorpion venom on ACh- and BRL 38227-induced relaxation were observed in the rabbit coronary artery. 7. Apamin (3.5 microM) was without effect on either the ACh- or BRL 38227-induced relaxation in the guinea-pig coronary artery. 8. In conclusion, the actions of BRL 38227 in coronary artery are compatible with its proposed effects on ATP-dependent K+ channels. In contrast, the results with ACh suggest that some step between the initial binding of ACh to endothelial muscarinic receptors and the final relaxation of the smooth muscle depends upon the opening of Ca(2+)-activated K+ channels.

1. Increases in intravascular pressure depolarize vascular smooth muscle cells. Based on the attenuating effects of Cl- channel antagonists, it has been suggested that swelling-activated Cl- channels may be integral to this response. Consequently, this study tested for the presence of a swelling-activated Cl- conductance in both intact rat cerebral arteries and isolated rat smooth muscle cells. 2. A 50 mosmol l-1 hyposmotic challenge (300 to 250 mosmol l-1) constricted rat cerebral arteries. This constriction contained all the salient features of a pressure-induced response including smooth muscle cell depolarization and a rise in intracellular Ca2+ that was blocked by voltage-operated Ca2+ channel antagonists. The hyposmotically induced depolarization was attenuated by DIDS (300 microM) and tamoxifen (1 microM), a response consistent with the presence of a swelling-activated Cl- conductance. 3. A swelling-activated current was identified in cerebral vascular smooth muscle cells. This current was sensitive to Cl- channel antagonists including DIDS (300 microM), tamoxifen (1 microM) and IAA-94 (100 microM). However, contrary to expectations, the reversal potential of this swelling-activated current shifted with the Na+ equilibrium potential and not the Cl- equilibrium potential, indicating that the swelling-activated current was carried by cations and not anions. The swelling-activated cation current was blocked by Gd3+, a cation channel antagonist. 4. Gd3+ also blocked both swelling- and pressure-induced depolarization of smooth muscle cells in intact cerebral arteries. 5. These findings suggest that swelling- and pressure-induced depolarization arise from the activation of a cation conductance. This current is inhibited by DIDS, tamoxifen, IAA-94 and gadolinium.

Elevated intracellular Ca2+([Ca2+]i) has been implicated in contractile and phenotypic changes in arterial smooth muscle during hypertension. This study examined the role of membrane potential and [Ca2+]iin altered gene expression in cerebral arteries of a rat (Dahl) genetic model of salt-sensitive hypertension. Cerebral arteries from hypertensive animals (Dahl salt-sensitive) exhibited a tonic membrane depolarization of ∼15 mV compared with normotensive (Dahl salt-resistant) animals. Consistent with this membrane depolarization, voltage-dependent K+currents were decreased in cerebral artery myocytes isolated from hypertensive animals. Arterial wall Ca2+was elevated in cerebral arteries from hypertensive animals, an effect reversed by diltiazem, a blocker of voltage-dependent Ca2+channels. This depolarization-induced increase in [Ca2+]iwas associated with increased activation of the transcription factor, cAMP response element binding protein, and increased expression of the immediate early gene c-fos, both of which are reversed by acute exposure to the voltage-dependent Ca2+channel blocker nisoldipine. This study provides the first information linking altered Ca2+handling to changes in gene expression in cerebral arteries during hypertension.