bluuebunny/crossref_metadata_2025 huggingface dataset:
P-450 epoxygenase and NO synthase inhibitors reduce cerebral blood flow response toN-methyl-d-aspartate
Authors: Bhardwaj Anish , Northington Frances J. , Carhuapoma Juan R. , Falck John R. , Harder David R. , Traystman Richard J. , Koehler Raymond C.
Published: December 2017

Epoxyeicosatrienoic acids are cerebral vasodilators produced in astrocytes by cytochrome P-450 epoxygenase activity. The P-450 inhibitor miconazole attenuates the increase in cerebral blood flow (CBF) elicited by glutamate. We evaluated whether epoxygenase activity is involved in the CBF response to activation of the N-methyl-d-aspartate (NMDA) receptor subtype by using two structurally distinct inhibitors, miconazole and N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH), a selective epoxygenase substrate inhibitor. Drugs were delivered locally through microdialysis probes in striata of anesthetized rats. Local CBF was measured by hydrogen clearance and compared with CBF in contralateral striatum receiving vehicle. Microdialysis perfusion of NMDA doubled CBF and increased nitric oxide (NO) production estimated by recovery of labeled citrulline in the dialysate during labeled arginine infusion. Perfusion of miconazole or MS-PPOH blocked the increase in CBF without decreasing citrulline recovery. Perfusion of Nω-nitro-l-arginine decreased baseline CBF and inhibited the CBF response to NMDA. Perfusion of MS-PPOH did not inhibit the CBF response to sodium nitroprusside. We conclude that both the P-450 epoxygenase and NO synthase pathways are involved in the local CBF response to NMDA receptor activation, and that the signaling pathway may be more complex than simply NO diffusion from neurons to vascular smooth muscle.
Cardioprotection by KATP channels in wild-type hearts and hearts overexpressing A1-adenosine receptors
Authors: Headrick John P. , Gauthier Naomi S. , Morrison Ray , Matherne G. Paul
Published: December 2017

We studied the role of mitochondrial ATP-sensitive K+(KATP) channels in modifying functional responses to 20 min global ischemia and 30 min reperfusion in wild-type mouse hearts and in hearts with ∼250-fold overexpression of functionally coupled A1-adenosine receptors (A1ARs). In wild-type hearts, time to onset of contracture (TOC) was 303 ± 24 s, with a peak contracture of 89 ± 5 mmHg. Diastolic pressure remained elevated at 52 ± 6 mmHg after reperfusion, and developed pressure recovered to 40 ± 6% of preischemia. A1AR overexpression markedly prolonged TOC to 517 ± 84 s, reduced contracture to 64 ± 6 mmHg, and improved recovery of diastolic (to 9 ± 4 mmHg) and developed pressure (to 82 ± 8%). 5-Hydroxydecanoate (5-HD; 100 μM), a mitochondrial KATPblocker, did not alter ischemic contracture in wild-type hearts, but increased diastolic pressure to 69 ± 8 mmHg and reduced developed pressure to 10 ± 5% during reperfusion. In transgenic hearts, 5-HD reduced TOC to 348 ± 18 s, increased postischemic contracture to 53 ± 4 mmHg, and reduced recovery of developed pressure to 22 ± 4%. In summary, these data are the first to demonstrate that endogenous activation of KATP channels improves tolerance to ischemia-reperfusion in murine myocardium. This functional protection occurs without modification of ischemic contracture. The data also support a role for mitochondrial KATP channel activation in the pronounced cardioprotection afforded by overexpression of myocardial A1ARs.
Relationship of molecular structure to the mechanism of lysophospholipid-induced smooth muscle cell proliferation
Authors: Chai Yuh-Cherng , Binion David G. , Chisolm Guy M.
Published: December 2017

We previously reported that oxidized low-density lipoprotein and one of its constituents, lysophosphatidylcholine (lysoPC), caused smooth muscle cell proliferation that was inhibitable by vitamin E and by a neutralizing antibody against basic fibroblast growth factor-2 (FGF-2). We now show that the mitogenic activity of lysolipids is highly dependent on structure. Phospholipids with palmitoyl fatty acid and phosphocholine induced DNA synthesis optimally. Shorter and longer fatty acids were significantly less potent, as were phosphoserine and phosphoethanolamine head groups. Structurally related phospholipids [platelet-activating factor (PAF) and lysoPAF] were also mitogens and acted via an analogous FGF-2-dependent, vitamin E-inhibitable mechanism. The mechanism of lysoPC stimulation was distinct from that of another phospholipid mitogen, lysophosphatidic acid (lysoPA), in that lysoPC stimulation was not pertussis toxin inhibitable. Furthermore, lysoPA stimulation was not inhibitable by vitamin E. Despite its distinct cellular pathway for stimulation, lysoPA also ultimately led to FGF-2 release. Our data show that specific structural attributes of lysoPC, PAF, and lysoPAF enable these agents to mediate smooth muscle cell release of FGF-2, which in turn stimulates proliferation.
Interactions of hemoglobin with hydrogen peroxide alters thiol levels and course of endothelial cell death
Authors: D'Agnillo Felice , Alayash Abdu I.
Published: December 2017

We investigated cellular injury and death induced by ultrapure human Hb (HbA0) and its diaspirin cross-linked derivative DBBF-Hb in normal and glutathione (GSH)-depleted bovine aortic endothelial cells subjected to hydrogen peroxide (H2O2). HbA0underwent extensive degradation and heme loss, whereas DBBF-Hb persisted longer in its ferryl (Fe4+) form. The formation of ferryl HbA0 or ferryl DBBF-Hb was associated with a significant decrease in endothelial cell GSH compared with the addition of H2O2 or Hbs alone. This effect was inhibited by catalase, but not by superoxide dismutase or deferoxamine mesylate. The presence of HbA0 and DBBF-Hb reduced H2O2-induced apoptosis, as measured by cell morphology, annexin V binding assay, and caspase inhibition, consistent with the ability to consume H2O2 in an enzyme-like fashion. However, the pattern of cell death and injury produced by HbA0 and DBBF-Hb appeared to be distinctly different among proteins as well as among cells with and without GSH. These findings may have important implications for the use of cell-free Hb as oxygen therapeutics in patients with coexisting pathologies who may lack antioxidant protective mechanisms.
Time and dose effect of transdermal nicotine on endothelial function
Authors: Miller Virginia M. , Clouse W. Darrin , Tonnessen Britt H. , Boston Umar S. , Severson Sandra R. , Bonde Sue , Rud Kevin S. , Hurt Richard D.
Published: December 2017

Nicotine patches are available as an over-the-counter medication for aid in smoking cessation. This study was designed to determine how nicotine patch therapy over time and dose ranges used in smoking cessation programs in humans affects endothelium-dependent relaxations. Dogs were treated with nicotine patches (11, 22, or 44 mg/day) for 2 and 5 wk. Circulating nicotine and oxidized products of nitric oxide (NOx) were measured. Coronary arteries were prepared for measurement of isometric force and aortic endothelial cells were prepared for measurement of mRNA or NO synthase (NOS) activity. Circulating nicotine increased with increasing concentrations of nicotine patches. After 5 wk of treatment with 22 mg/day patches, circulating NOx was reduced but NOS activity was increased. NOS mRNA was similar among groups. Only after 5 wk of treatment with 22 mg/day patches were endothelium-dependent relaxations reduced to α2-adrenergic agonists, ADP, and the calcium ionophore A-23187. These results suggest a time and biphasic dose effect of nicotine treatment on endothelium-dependent responses that may be related to bioavailability of NO. This complex relationship of duration and dose of nicotine treatment may explain, in part, discrepancies in effects of nicotine on endothelium-dependent responses.
Regulation of a voltage-sensitive release mechanism by Ca2+-calmodulin-dependent kinase in cardiac myocytes
Authors: Zhu Jiequan , Ferrier Gregory R.
Published: December 2017

A role for Ca2+-calmodulin-dependent kinase (CamK) in regulation of the voltage-sensitive release mechanism (VSRM) was investigated in guinea pig ventricular myocytes. Voltage clamp was used to separate the VSRM from Ca2+-induced Ca2+release (CICR). VSRM contractions and Ca2+transients were absent in cells dialyzed with standard pipette solution but present when 2–5 μM calmodulin was included. Effects of calmodulin were blocked by KN-62 (CamK inhibitor), but not H-89, a protein kinase A (PKA) inhibitor. Ca2+current and caffeine contractures were not affected by calmodulin. Transient-voltage relations were bell-shaped without calmodulin, but they were sigmoidal and typical of the VSRM with calmodulin. Contractions with calmodulin exhibited inactivation typical of the VSRM. These contractions were inhibited by rapid application of 200 μM of tetracaine, but not 100 μM of Cd2+, whereas CICR was inhibited by Cd2+but not tetracaine. In undialyzed myocytes (high-resistance microelectrodes), KN-62 or H-89 each reduced amplitudes of VSRM contractions by ∼50%, but together they decreased VSRM contractions by 93%. Thus VSRM is facilitated by CamK or PKA, and both pathways regulate the VSRM in undialyzed cells.
Autocrine thrombospondin partially mediates TGF-β1- induced proliferation of vascular smooth muscle cells
Authors: Sajid Mansoor , Lele Manjiri , Stouffer George A.
Published: December 2017

Transforming growth factor (TGF)-β1 has been implicated in vascular healing responses after mechanical injury. Using cultured rat aortic smooth muscle cells (RASMC), we examined the hypothesis that production and secretion of thrombospondin (TSP) contributes to TGF-β1-induced proliferation. We found that TGF-β1 enhanced production and secretion of TSP, with peak levels of secreted TSP observed 24 h after treatment. RASMC treated with TGF-β1 secreted a mitogenic activity that was transferable in conditioned media and partially inhibited by C6.7, a monoclonal anti-TSP antibody. Exogenous TSP stimulated a proliferative response, with maximal [3H]thymidine incorporation occurring 24 h earlier than maximal [3H]thymidine incorporation in response to TGF-β1-treatment. Pretreatment with C6.7 or polyclonal anti-TSP neutralizing antibodies inhibited TGF-β1-induced proliferation of RASMC. Proliferative responses to TGF-β1 were also inhibited by pretreatment with an anti-β3integrin monoclonal blocking antibody (F11), RGD peptides, and the anti-αvβ3disintegrin echistatin. Treatment with TSP and TGF-β1 increased c-Jun NH2-terminal kinase (JNK)1 activity, with peak effects observed at 15 min and 4 h, respectively. Treatment with C6.7 or F11 inhibited TGF-β-induced activation of JNK1. In summary, these studies support the hypothesis that TGF-β-induced JNK1 activation and proliferation of RASMC require secretion of TSP and ligation of αvβ3-integrins.
Leukocyte and endothelial cell adhesion molecules in a chronic murine model of myocardial reperfusion injury
Authors: Jones Steven P. , Trocha Steven D. , Strange Micah B. , Granger D. Neil , Kevil Christopher G. , Bullard Daniel C. , Lefer David J.
Published: December 2017

Expression of endothelial and leukocyte cell adhesion molecules is a principal determinant of polymorphonuclear neutrophil (PMN) recruitment during inflammation. It has been demonstrated that pharmacological inhibition of these molecules can attenuate PMN influx and subsequent tissue injury. We determined the temporal expression of α-granule membrane protein-40 (P-selectin), endothelial leukocyte adhesion molecule 1 (E-selectin), and intercellular cell adhesion molecule 1 (ICAM-1) after coronary artery occlusion and up to 3 days of reperfusion. The expression of all of these cell adhesion molecules peaked around 24 h of reperfusion. We determined the extent to which these molecules contribute to PMN infiltration by utilizing mice deficient (−/−) in P-selectin, E-selectin, ICAM-1, and CD18. Each group underwent 30 min of in vivo, regional, left anterior descending (LAD) coronary artery ischemia and 24 h of reperfusion. PMN accumulation in the ischemic-reperfused (I/R) zone was assessed using histological techniques. Deficiencies of P-selectin, E-selectin, ICAM-1, or CD18 resulted in significant ( P < 0.05) attenuation of PMN infiltration into the I/R myocardium (MI/R). In addition, P-selectin, E-selectin, ICAM-1, and CD18 −/− mice exhibited significantly ( P < 0.05) smaller areas of necrosis after MI/R compared with wild-type mice. These data demonstrate that MI/R induces coronary vascular expression of P-selectin, E-selectin, and ICAM-1 in mice. Furthermore, genetic deficiency of P-selectin, E-selectin, ICAM-1, or CD18 attenuates PMN sequestration and myocardial injury after in vivo MI/R. We conclude that P-selectin, E-selectin, ICAM-1, and CD18 are involved in the pathogenesis of MI/R injury in mice.
Acute effects of 17β-estradiol on ventricular and vascular hemodynamics in postmenopausal women
Authors: Hayward Christopher S. , Kalnins Wally V. , Kelly Raymond P.
Published: December 2017

Because premenopausal women have lower cardiovascular morbidity than postmenopausal women, it has been proposed that estrogen may have a protective role. Estrogen is involved in smooth muscle relaxation both through its specific receptor as well as through calcium channel blockade. This study examined the acute effect of estradiol on invasive cardiovascular hemodynamics in 18 postmenopausal women (age 62.6 ± 7.6 years, means ± SD). The effect of estradiol on left ventricular chamber performance was studied in 9 women using simultaneous left ventricular pressure-volume recordings. In a further group of 9 women, the acute effect of estradiol on arterial function was assessed using input impedance (derived from simultaneous aortic pressure and flow recordings), pressure waveform analysis, and pulse wave velocity. After 2 mg micronized 17β-estradiol was administered, serum estradiol levels increased from 50.9 ± 21.9 to 3,190 ± 2,216 pmol/l, P < 0.0001. There was no effect of estradiol on either left ventricular inotropic or lusitropic function. There was no acute effect of estradiol on arterial impedance, reflection coefficient, augmentation index, or pulse wave velocity. There was a trend to decreased heart rate and cardiac output in both groups of 9 women. Because heart rate and cardiac output were common to both hemodynamic data sets, results for these parameters were pooled. Across all 18 women, there was a small but significant decrease in heart rate (69.2 ± 10.4 vs. 67.2 ± 9.9 beats/min, P = 0.02), as well as a significant decrease in cardiac output (4.82 ± 1.77 vs. 4.17 ± 1.56 l/min, P = 0.002). Despite achieving supraphysiological serum levels, this study found no significant effect of acute 17β-estradiol on ventricular or large artery function.
Microvascular flow and tissue Po 2 in skeletal muscle of chronic reduced renal mass hypertensive rats
Authors: Lombard Julian H. , Frisbee Jefferson C. , Greene Andrew S. , Hudetz Antal G. , Roman Richard J. , Tonellato Peter J.
Published: December 2017

This study determined whether arteriolar blood flow, capillary red blood cell (RBC) velocity, capillary hematocrit (Hctcap), and tissue Po 2 are altered in cremaster muscles of rats with chronic reduced renal mass hypertension (RRM-HT) relative to normotensive rats on high- or low-salt (NT-HS vs. NT-LS) diet. The blood flow in first- through third-order arterioles was not different between NT and HT rats, either at rest or during maximal relaxation of the vessels with 10−4 M adenosine. Capillary RBC velocity was similar between the groups at rest but was elevated in RRM-HT and NT-HS rats during adenosine superfusion. Hctcap was reduced at rest in RRM-HT and NT-HS rats compared with NT-LS and was reduced in RRM-HT rats during adenosine-induced dilation. Tissue Po 2 was reduced in RRM-HT and NT-HS rats compared with NT-LS rats during control conditions and was lower in RRM-HT than in NT-LS rats during adenosine-induced dilation. These results indicate that both RRM-HT and chronic exposure of normotensive rats to a high-salt diet lead to reduced tissue oxygenation, despite the maintenance of normal arteriolar blood flow.
Biphasic response of cardiac NO synthase isoforms to ischemic preconditioning in conscious rabbits
Authors: Xuan Yu-Ting , Tang Xian-Liang , Qiu Yumin , Banerjee Supratim , Takano Hitoshi , Han Hui , Bolli Roberto
Published: December 2017

In conscious rabbits, a sequence of six 4-min coronary occlusion/4-min reperfusion cycles, which elicits late preconditioning (PC), caused rapid activation of calcium-dependent nitric oxide (NO) synthase (NOS) [cNOS; endothelial NOS (eNOS) and/or neuronal NOS (nNOS)], whereas calcium-independent NOS [inducible NOS (iNOS)] activity remained unchanged. The enhanced cNOS activity was associated with increased myocardial levels of NO2and/or NO3(NOx). Twenty-four hours after ischemic PC was induced, the opposite pattern was observed, i.e., there was a pronounced increase in cytosolic iNOS activity but no change in cNOS activity. The initial burst of ischemia-induced cNOS activity was not affected by pretreatment with the antioxidant N-2-mercaptopropionyl glycine (MPG), the protein kinase C (PKC) inhibitor chelerythrine, or the tyrosine kinase inhibitor lavendustin A, indicating that it is independent of the generation of oxidant species and the activation of PKC and tyrosine kinases. In contrast, the delayed upregulation of iNOS 24 h after PC was prevented by pretreatment with Nω-nitro-l-arginine, MPG, or chelerythrine before the PC ischemia, indicating that it is triggered by a signaling mechanism that involves the generation of NO, the formation of oxidant species, and the activation of PKC. Taken together, these results demonstrate that, in conscious animals, ischemic PC elicits a biphasic response in cardiac NOS activity, i.e., an immediate activation of cNOS (most likely eNOS) followed 24 h later by a delayed upregulation of iNOS. To our knowledge, this is the first study to directly measure NOS activity after brief myocardial ischemia in vivo. In conjunction with previous functional studies, the data support a distinctive role of NOS isoforms in late PC, with eNOS serving as the trigger on day 1 and iNOS as the mediator on day 2.
Effects of hemoglobin on heme oxygenase gene expression and viability of cultured smooth muscle cells
Authors: Marton Linda S. , Wang Xiaoyu , Kowalczuk Andrew , Zhang Zhen-Du , Windmeyer Emily , Macdonald R. Loch
Published: December 2017

Ferrous Hb contributes to cerebral vasospasm after subarachnoid hemorrhage, although the mechanisms involved are uncertain. The hypothesis that cytotoxic effects of ferrous Hb on smooth muscle cells contribute to vasospasm was assessed. Cultured rat basilar artery smooth muscle cells were exposed to pure Hb, dog erythrocyte hemolysate, or Hb breakdown products; and heme oxygenase (HO-1 and HO-2) and ferritin mRNA and protein were measured. Cytotoxicity was assessed by lactate dehydrogenase release and fluorescence assays. Pure Hb or hemolysate caused dose- and time-dependent increases in HO-1 mRNA and protein. Hemin was the component of Hb that increased HO-1 mRNA. Cycloheximide inhibited the increase in HO-1 mRNA in response to hemin. Ferritin protein heavy chain but not mRNA increased upon exposure of cells to Hb. Hemin and ferric but not ferrous Hb were toxic to smooth muscle cells. Toxicity was increased by exposure to Hb plus tin protoporphyrin IX. In conclusion, exposure of smooth muscle cells to Hb induces HO-1 mRNA and protein through pathways that involve new protein synthesis. Hemin is the component of Hb that induces HO-1. Hemin and ferric but not ferrous Hb are toxic.
Assessing baroreflex gain from spontaneous variability in conscious dogs: role of causality and respiration
Authors: Porta A. , Baselli G. , Rimoldi O. , Malliani A. , Pagani M.
Published: December 2017

A double exogenous autoregressive (XXAR) causal parametric model was used to estimate the baroreflex gain (αXXAR) from spontaneous R-R interval and systolic arterial pressure (SAP) variabilities in conscious dogs. This model takes into account 1) effects of current and past SAP variations on the R-R interval (i.e., baroreflex-mediated influences), 2) specific perturbations affecting R-R interval independently of baroreflex circuit (e.g., rhythmic neural inputs modulating R-R interval independently of SAP at frequencies slower than respiration), and 3) influences of respiration-related sources acting independently of baroreflex pathway (e.g., rhythmic neural inputs modulating R-R interval independently of SAP at respiratory rate, including the effect of stimulation of low-pressure receptors). Under control conditions, αXXAR = 14.7 ± 7.2 ms/mmHg. It decreases after nitroglycerine infusion and coronary artery occlusion, even though the decrease is significant only after nitroglycerine, and it is completely abolished by total arterial baroreceptor denervation. Moreover, αXXAR is comparable to or significantly smaller than (depending on the experimental condition) the baroreflex gains derived from sequence, power spectrum [at low frequency (LF) and high frequency (HF)], and cross-spectrum (at LF and HF) analyses and from less complex causal parametric models, thus demonstrating that simpler estimates may be biased by the contemporaneous presence of regulatory mechanisms other than baroreflex mechanisms.
Transmural difference in coronary arteriolar dilation to adenosine: effect of luminal pressure and KATP channels
Authors: Zhang Cuihua , Hein Travis W. , Kuo Lih
Published: December 2017

Coronary blood flow in the subendocardium is preferentially increased by adenosine but is redistributed to the subepicardium during ischemia in association with coronary pressure reduction. The mechanism for this flow redistribution remains unclear. Since adenosine is released during ischemia, it is possible that the coronary microcirculation exhibits a transmural difference in vasomotor responsiveness to adenosine at various intraluminal pressures. Although the ATP-sensitive K+ (KATP) channel has been shown to be involved in coronary arteriolar dilation to adenosine, its role in the transmural adenosine response remains elusive. To address these issues, pig subepicardial and subendocardial arterioles (60–120 μm) were isolated, cannulated, and pressurized to 20, 40, 60, or 80 cmH2O without flow for in vitro study. At each of these pressures, vessels developed basal tone and dilated concentration dependently to adenosine and the KATP channel opener pinacidil. Subepicardial and subendocardial arterioles dilated equally to adenosine and pinacidil at 60 and 80 cmH2O luminal pressure. At lower luminal pressures (i.e., 20 and 40 cmH2O), vasodilation in both vessel types was enhanced. Enhanced vasodilatory responses were not affected by removal of endothelium but were abolished by the KATP channel inhibitor glibenclamide. In a manner similar to reducing pressure, a subthreshold dose of pinacidil potentiated vasodilation to adenosine. In contrast to adenosine, dilation of coronary arterioles to sodium nitroprusside was independent of pressure changes. These results indicate that coronary microvascular dilation to adenosine is enhanced at lower intraluminal pressures by selective activation of smooth muscle KATP channels. Since microvascular pressure has been shown to be consistently lower in the subendocardium than in the subepicardium, it is likely that the inherent pressure gradient in the coronary microcirculation across the ventricular wall may be an important determinant of transmural flow in vivo during resting conditions or under metabolic stress with adenosine release.
Effect of combined KATP channel activation and Na+/H+ exchange inhibition on infarct size in rabbits
Authors: Hale Sharon L. , Kloner Robert A.
Published: December 2017

We tested if combining treatment with cariporide, an Na+/H+ exchange inhibitor, and diazoxide, a mitochondrial ATP-sensitive K+ (KATP) channel opener, would reduce myocardial infarct size (IS) to a greater extent than either intervention alone. Four groups of rabbits were studied ( n = 10 each): cariporide (0.3 mg/kg), diazoxide (10 mg/kg), both drugs, and saline control, given 15 min before a 30-min coronary artery occlusion and 3 h reperfusion. IS in controls comprised 47 ± 6% of the risk region. Cariporide reduced IS by 55% compared with control (21 ± 3%), but diazoxide did not significantly reduce IS compared with controls (37 ± 6%). Combined treatment resulted in an IS of 18 ± 5%. Also we determined that diazoxide did not potentiate a subthreshold dose of cariporide nor did a mitochondrial KATP channel blocker, 5-hydroxydecanoate (5-HD), prevent cariporide from reducing IS. Thus cariporide reduced necrosis by >50% in this model, both in the presence and absence of KATP channel blockade. There was no significant difference in IS reduction between the group receiving cariporide alone and the group receiving combined treatment. Because the effect of cariporide was not blocked by 5-HD, it is unlikely that KATP channels play a role as an end effector in cariporide's mechanism.
Role of inwardly rectifying K+ channels in K+-induced cerebral vasodilatation in vivo
Authors: Chrissobolis Sophocles , Ziogas James , Chu Yi , Faraci Frank M. , Sobey Christopher G.
Published: December 2017

We tested whether activation of inwardly rectifying K+ (Kir) channels, Na+-K+-ATPase, or nitric oxide synthase (NOS) play a role in K+-induced dilatation of the rat basilar artery in vivo. When cerebrospinal fluid [K+] was elevated from 3 to 5, 10, 15, 20, and 30 mM, a reproducible concentration-dependent vasodilator response was elicited (change in diameter = 9 ± 1, 27 ± 4, 35 ± 4, 43 ± 12, and 47 ± 16%, respectively). Responses to K+ were inhibited by ∼50% by the Kir channel inhibitor BaCl2 (30 and 100 μM). In contrast, neither ouabain (1–100 μM, a Na+-K+-ATPase inhibitor) nor N G-nitro-l-arginine (30 μM, a NOS inhibitor) had any effect on K+-induced vasodilatation. These concentrations of K+ also hyperpolarized smooth muscle in isolated segments of basilar artery, and these hyperpolarizations were virtually abolished by 30 μM BaCl2. RT-PCR experiments confirmed the presence of mRNA for Kir2.1 in the basilar artery. Thus K+-induced dilatation of the basilar artery in vivo appears to partly involve hyperpolarization mediated by Kir channel activity and possibly another mechanism that does not involve hyperpolarization, activation of Na+-K+-ATPase, or NOS.
NADH/NAD redox state of cytoplasmic glycolytic compartments in vascular smooth muscle
Authors: Barron John T. , Gu Liping , Parrillo Joseph E.
Published: December 2017

The cytoplasmic NADH/NAD redox potential affects energy metabolism and contractile reactivity of vascular smooth muscle. NADH/NAD redox state in the cytosol is predominately determined by glycolysis, which in smooth muscle is separated into two functionally independent cytoplasmic compartments, one of which fuels the activity of Na+-K+-ATPase. We examined the effect of varying the glycolytic compartments on cystosolic NADH/NAD redox state. Inhibition of Na+-K+-ATPase by 10 μM ouabain resulted in decreased glycolysis and lactate production. Despite this, intracellular concentrations of the glycolytic metabolite redox couples of lactate/pyruvate and glycerol-3-phosphate/dihydroxyacetone phosphate (thus NADH/NAD) and the cytoplasmic redox state were unchanged. The constant concentration of the metabolite redox couples and redox potential was attributed to 1) decreased efflux of lactate and pyruvate due to decreased activity of monocarboxylate B-H+ transporter secondary to decreased availability of H+ for cotransport and 2) increased uptake of lactate (and perhaps pyruvate) from the extracellular space, probably mediated by the monocarboxylate-H+ transporter, which was specifically linked to reduced activity of Na+-K+-ATPase. We concluded that redox potentials of the two glycolytic compartments of the cytosol maintain equilibrium and that the cytoplasmic NADH/NAD redox potential remains constant in the steady state despite varying glycolytic flux in the cytosolic compartment for Na+-K+-ATPase.
Renin-angiotensin system inhibition on noradrenergic nerve terminal function in pacing-induced heart failure
Authors: Kawai Hiroya , Stevens Suzanne Y. , Liang Chang-Seng
Published: December 2017

Chronic angiotensin-converting enzyme (ACE) inhibition has been shown to improve cardiac sympathetic nerve terminal function in heart failure. To determine whether similar effects could be produced by angiotensin II AT1 receptor blockade, we administered the ACE inhibitor quinapril, angiotensin II AT1 receptor blocker losartan, or both agents together, to rabbits with pacing-induced heart failure. Chronic rapid pacing produced left ventricular dilation and decline of fractional shortening, increased plasma norepinephrine (NE), and caused reductions of myocardial NE uptake activity, NE histofluorescence profile, and tyrosine hydroxylase immunostained profile. Administration of quinapril or losartan retarded the progression of left ventricular dysfunction and attenuated cardiac sympathetic nerve terminal abnormalities in heart failure. Quinapril and losartan together produced greater effects than either agent alone. The effect of renin-angiotensin system inhibition on improvement of left ventricular function and remodeling, however, was not sustained. Our results suggest that the effects of ACE inhibitors are mediated via the reduction of angiotensin II and that angiotensin II plays a pivotal role in modulating cardiac sympathetic nerve terminal function during development of heart failure. The combined effect of ACE inhibition and angiotensin II AT1 receptor blockade on cardiac sympathetic nerve terminal dysfunction may contribute to the beneficial effects on cardiac function in heart failure.
Design and construction of a uniaxial cell stretcher
Authors: Yost Michael J. , Simpson David , Wrona Kimberly , Ridley Stephen , Ploehn Harry J. , Borg Thomas K. , Terracio Louis
Published: December 2017

In vitro mechanical cell stimulators are used for the study of the effect of mechanical stimulation on anchorage-dependent cells. We developed a new mechanical cell stimulator, which uses stepper motor technology and computer control to achieve a high degree of accuracy and repeatability. This device also uses high-performance plastic components that have been shown to be noncytotoxic, dimensionally stable, and resistant to chemical degradation from common culture laboratory chemicals. We show that treatment with glow discharge for 25 s at 20 mA is sufficient to modify the surface of the rubber to allow proper adhesion for polymerization of aligned collagen. We show through finite element analysis that the middle area of the membrane, away from the clamped ends, is predictable, homogeneous, and has negligible shear strain. To test the efficacy of the mechanical stretch, we examined the effect of mechanical stimulation on the production of β1-integrin by neonatal rat cardiac fibroblasts. Mechanical stimulation was tested in the range of 0–12% stretch and 0–10-cycles/min stretch frequency. The fibroblasts respond with an increase in β1-integrin at 3% stretch and a decrease at 6 and 12% stretch. Stretch frequency was found to not significantly effect the concentration of β1-integrin. These studies yield a new and improved mechanical cell stimulator and demonstrate that mechanical stimulation has an effect on the expression of β1-integrin.
Stable labeled microspheres to measure perfusion: validation of a neutron activation assay technique
Authors: Reinhardt Christopher P. , Dalhberg Seth , Tries Mark A. , Marcel Robin , Leppo Jeffery A.
Published: December 2017

Neutron activation is an accurate analytic method in which trace quantities of isotopes of interest in a sample are activated and the emitted radiation is measured with high-resolution detection equipment. This study demonstrates the application of neutron activation for the measurement of myocardial perfusion using stable isotopically labeled microspheres. Stable labeled and standard radiolabeled microspheres (15 μm) were coinjected in an in vivo rabbit model of myocardial ischemia and reperfusion. Radiolabeled microspheres were detected with a standard gamma-well counter, and stable labeled microspheres were detected with a high-resolution Ge detection after neutron activation of the myocardial and reference blood samples. Regional myocardial blood flow was calculated from the deposition of radiolabeled and stable labeled microspheres. Both sets of microspheres gave similar measurements of regional myocardial blood flow over a wide range of flow with a high linear correlation ( r = 0.95–0.99). Neutron activation is capable of detecting a single microsphere in an intact myocardial sample while providing simultaneous quantitative measurements of multiple isotope labels. This high sensitivity and capability for measuring perfusion in intact tissue are advantages over other techniques, such as optical detection of microspheres. Neutron activation also can provide an effective method for reducing the production of low-level radioactive waste generated from biomedical research. Further applications of neutron activation offer the potential for measuring other stable labeled compounds, such as fatty acids and growth factors, in conjunction with microsphere measured flow, providing the capability for simultaneous measurement of regional metabolism and perfusion.
Progression from hypertrophic to dilated cardiomyopathy in mice that express a mutant myosin transgene
Authors: Freeman Kalev , Colon-Rivera Cynthia , Olsson M. Charlotte , Moore Russell L. , Weinberger Howard D. , Grupp Ingrid L. , Vikstrom Karen L. , Iaccarino Guido , Koch Walter J. , Leinwand Leslie A.
Published: December 2017

A mouse model of hypertrophic cardiomyopathy (HCM) was created by expression of a cardiac α-myosin transgene including the R403Q mutation and a deletion of a segment of the actin-binding domain. HCM mice show early histopathology and hypertrophy, with progressive hypertrophy in females and ventricular dilation in older males. To test the hypothesis that dilated cardiomyopathy (DCM) is part of the pathological spectrum of HCM, we studied chamber morphology, exercise tolerance, hemodynamics, isolated heart function, adrenergic sensitivity, and embryonic gene expression in 8- to 11-mo-old male transgenic animals. Significantly impaired exercise tolerance and both systolic and diastolic dysfunction were seen in vivo. Contraction and relaxation parameters of isolated hearts were also decreased, and lusitropic responsiveness to the β-adrenergic agonist isoproterenol was modestly reduced. Myocardial levels of the G protein-coupled β-adrenergic receptor kinase 1 (β-ARK1) were increased by more than twofold over controls, and total β-ARK1 activity was also significantly elevated. Induction of fetal gene expression was also observed in transgenic hearts. We conclude that transgenic male animals have undergone cardiac decompensation resulting in a DCM phenotype. This supports the idea that HCM and DCM may be part of a pathological continuum rather than independent diseases.
Role of p38 MAP kinase in endothelial cell alignment induced by fluid shear stress
Authors: Azuma Nobuyoshi , Akasaka Nobuyuki , Kito Hiroyuki , Ikeda Masataka , Gahtan Vivian , Sasajima Tadahiro , Sumpio Bauer E.
Published: December 2017

The p38/mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MAPKAP kinase 2)/heat shock protein (HSP)25/27 pathway is thought to play a critical role in actin dynamics. In the present study, we examined whether p38 was involved in the morphological changes seen in endothelial cells (EC) exposed to shear stress. Cultured bovine aortic EC were subjected to 14 dyn/cm2laminar steady shear stress. Peak activation of p38, MAPKAP kinase 2, and HSP25 were sixfold at 5 min, sixfold at 5 min, and threefold at 30 min compared with static control, respectively. SB-203580 (1 μM), a specific inhibitor of p38, abolished the activation of MAPKAP kinase 2 and HSP25 as well as EC elongation and alignment in the direction of flow elicited by shear stress. The mean orientation angle of cells subjected to shear without SB-203580, with SB-203580, or static control were 17, 50, and 43°, respectively ( P < 0.05). EC transfected with the dominant negative mutant of p38-α aligned randomly with no stress fiber formation despite exposure to shear stress. These data suggests that the pathway of p38/MAPKAP kinase 2/HSP25/27 is activated in response to shear stress, and this pathway plays an important role in morphological changes induced by shear stress.
Vagosympathetic interactions in ischemia-induced myocardial norepinephrine and acetylcholine release
Authors: Kawada Toru , Yamazaki Toji , Akiyama Tsuyoshi , Inagaki Masashi , Shishido Toshiaki , Zheng Can , Yanagiya Yusuke , Sugimachi Masaru , Sunagawa Kenji
Published: December 2017

To elucidate the pathophysiological roles of vagosympathetic interactions in ischemia-induced myocardial norepinephrine (NE) and acetylcholine (ACh) release, we measured myocardial interstitial NE and ACh levels in response to a left anterior descending coronary occlusion in the following groups of anesthetized cats: intact autonomic innervation (INT, n = 7); vagotomy (VX, n = 6); local administration of atropine (Atro, n = 6); transection of the stellate ganglia (TSG, n = 5); local administration of phentolamine (Phen, n = 6); and combined vagotomy and transection of the stellate ganglia (VX+TSG, n = 5). The maximum NE release was enhanced in the VX group (141 ± 30 nmol/l, means ± SE, P < 0.05) compared with the INT group (61 ± 12 nmol/l). Neither the Atro (50 ± 24 nmol/l) nor VX+TSG groups (84 ± 25 nmol/l) showed enhanced NE release. The maximum ACh release was unaltered in the TSG and Phen groups compared with the INT group (19 ± 4, 18 ± 4, and 13 ± 3 nmol/l, respectively). These findings indicate that the cardiac vagal afferent but not efferent activity reduced the ischemia-induced myocardial NE release. In contrast, the cardiac sympathetic afferent and efferent activities played little role in the ischemia-induced myocardial ACh release.
Dual roles of mitochondrial KATP channels in diazoxide-mediated protection in isolated rabbit hearts
Authors: Wang Sheng , Cone James , Liu Yongge
Published: December 2017

Whether the mitochondrial ATP-dependent potassium (mKATP) channel is the trigger or the mediator of cardioprotection is controversial. We investigated the critical time sequences of mKATP channel opening for cardioprotection in isolated rabbit hearts. Pretreatment with diazoxide (100 μM), a selective mKATP channel opener, for 5 min followed by 10 min washout before the 30-min ischemia and 2-h reperfusion significantly reduced infarct size (9 ± 3 vs. 35 ± 3% in control), indicating a role of mKATP channels as a trigger of protection. The protection was blocked by coadministration of the L-type Ca2+ channel blockers nifedipine (100 nM) or 5-hydroxydecanoic acid (5-HD; 50 μM) or by the protein kinase C (PKC) inhibitor chelerythrine (5 μM). The protection of diazoxide was not blocked by 50 μM 5-HD but was blocked by 200 μM 5-HD or 10 μM glybenclamide administrated 5 min before and throughout the 30 min of ischemia, indicating a role of mKATP opening as a mediator of protection. Giving diazoxide throughout the 30 min of ischemia also protected the heart, and the protection was not blocked by chelerythrine. Nifedipine did not affect the ability of diazoxide to open mKATP channels assessed by mitochondrial redox state. In electrically stimulated rabbit ventricular myocytes, diazoxide significantly increased Ca2+ transient but had no effect on L-type Ca2+ currents. Our results suggest that opening of mKATP channels can trigger cardioprotection. The trigger phase may be induced by elevation of intracellular Ca2+ and activation of PKC. During the lethal ischemia, mKATPchannel opening mediates the protection, independent of PKC, by yet unknown mechanisms.
Inducible regulation of human brain natriuretic peptide promoter in transgenic mice
Authors: He Quan , Wang Ding , Yang Xiao-Ping , Carretero Oscar A. , LaPointe Margot C.
Published: December 2017

Studies have shown that brain natriuretic peptide (BNP) gene expression is rapidly induced in the infarcted heart and that plasma BNP levels reflect the degree of left ventricular dysfunction. Our previous in vitro work using transiently transfected neonatal rat cardiac myocytes has shown that the human BNP (hBNP) promoter, in particular a region extending from −127 to −40 relative to the start site of transcription, is more active in cardiac myocytes than in fibroblasts. To study tissue-specific and transcriptional regulation of the hBNP gene in vivo, we generated transgenic mice containing the proximal hBNP promoter (−408 to +100) coupled to a luciferase reporter gene. In four lines of transgenic mice, luciferase activity was ∼33- to 100-fold higher in the heart than in other tissues, including the whole brain. To test whether the transgene responded to a pathophysiological stimulus, we induced infarction by coronary artery ligation. Luciferase activity was fivefold higher in the infarcted region of the left ventricle at 48 h than in sham-operated animals and remained elevated for 4 wk. Endogenous BNP mRNA was similarly increased in the infarcted hearts of a separate group of mice. We conclude that 1) the proximal 408-bp region of the hBNP promoter confers cardiac-specific expression and 2) myocardial infarction activates the proximal hBNP promoter in vivo. These data suggest that we have a valid model for the study of basal and inducible regulation of the hBNP gene in vivo.
Inhibition of p38 MAPK α/β reduces ischemic injury and does not block protective effects of preconditioning
Authors: Schneider Sharron , Chen Weina , Hou Janet , Steenbergen Charles , Murphy Elizabeth
Published: December 2017

We examined the effect of inhibition of p38 mitogen-activated protein kinase (MAPK) α/β during ischemia and preconditioning by using the inhibitor SB-202190. Isolated rat hearts were perfused with Krebs-Henseleit buffer, while left ventricular developed pressure (LVDP) and31P nuclear magnetic resonance spectra were acquired continuously. After 20 min of ischemia and 25 min of reperfusion, recovery of LVDP in untreated hearts was 32 ± 4%, whereas hearts treated with SB-202190 5 min before ischemia recovered 59 ± 7% of their pretreatment LVDP. Preconditioning improved functional recovery to 65 ± 5%, which was unaffected by SB-202190 treatment, added either throughout the preconditioning protocol (56 ± 5% recovery) or during the final reperfusion period of preconditioning (71 ± 11% recovery). Necrosis was assessed after 40 min of ischemia and 2 h of reperfusion using 2,3,5-triphenyltetrazolium chloride (TTC) staining and creatine kinase release. The untreated group had 54 ± 8% necrotic myocardium, whereas the SB-202190-treated group had 32 ± 7% and the preconditioned group had 21 ± 4% necrotic tissue by TTC staining.
Decreased NADH dehydrogenase and ubiquinol-cytochromecoxidoreductase in peripheral arterial disease
Authors: Brass Eric P. , Hiatt William R. , Gardner Andrew W. , Hoppel Charles L.
Published: December 2017

Peripheral arterial disease (PAD) is associated with muscle metabolic changes that may contribute to the disability in these patients. However, the biochemical defects in PAD have not been identified. The present study was undertaken to test the hypothesis that PAD is associated with specific defects in skeletal muscle electron transport chain activity. Seventeen patients with PAD and nine age-matched controls underwent gastrocnemius muscle biopsies. There were no differences in the mitochondrial content per gram of skeletal muscle as assessed by citrate synthase activity between the PAD patients and the control subjects. Skeletal muscle NADH dehydrogenase activity was decreased by 27% compared with controls when expressed per unit of citrate synthase activity. Expression of enzyme activities normalized to cytochrome c-oxygen oxidoreductase activity confirmed a 26% decrease in NADH dehydrogenase activity and also demonstrated a 38% decrease in ubiquinol-cytochrome c oxidoreductase activity. Thus PAD is associated with specific changes in muscle mitochondrial electron transport chain activities characterized by relative decreases in NADH dehydrogenase and ubiquinol-cytochrome c oxidoreductase activities, which may contribute to the metabolic abnormalities and decreased exercise performance in these patients.
Arteriolar nitric oxide concentration is decreased during hyperglycemia-induced βII PKC activation
Authors: Bohlen H. Glenn , Nase Geoffrey P.
Published: December 2017

βII protein kinase C (βPKC) is activated during acute and chronic hyperglycemia and may alter endothelial cell function. We determined whether blockade of βPKC protected in vivo endothelial formation of NO, as measured with NO-sensitive microelectrodes in the rat intestinal vasculature. NaCl hyperosmolarity, a specific endothelial stimulus to increase NO formation, caused ∼20% arteriolar vasodilation and ∼30% increase in NO concentration ([NO]). After topical 300 mg/dl hyperglycemia for 45 min, both responses were all but abolished. In comparison, pretreatment with LY-333531, a specific βPKC inhibitor, maintained vasodilation and [NO] responses to NaCl hyperosmolarity after hyperglycemia. The βPKC inhibitor alone had no significant effects on resting diameter or [NO] or their responses to NaCl hyperosmolarity. In separate rats, after topical hyperglycemia had suppressed dilation to ACh, LY-333531 restored ∼70% of the dilatory response. These data demonstrated that activation of βPKC during acute hyperglycemia depressed in vivo endothelial formation of NO at rest and during stimulation. This abnormality can be minimized by inhibition of βPKC before hyperglycemia and can be substantially reversed by PKC inhibition after hyperglycemia-induced abnormalities have occurred.
Effects of acupuncture on radial artery hemodynamics: controlled trials in sensitized and naive subjects
Authors: Boutouyrie Pierre , Corvisier Robert , Azizi Michel , Lemoine Dominique , Laloux Brigitte , Hallouin Marie-Charlotte , Laurent Stéphane
Published: December 2017

Palpation of the radial pulses is an important technique in traditional Chinese medicine. Two double-blind randomized trials of the effects of real and sham acupuncture on radial artery hemodynamics were conducted in 19 patients regularly exposed to acupuncture (sensitized subjects) and in 8 healthy subjects devoid of previous exposure (naive subjects), respectively. Radial artery diameter and pulse waveform were measured with a high-resolution echotracking system and aplanation tonometry, respectively, before and during a 20-min acupuncture period. In sensitized patients, arterial diameter significantly increased during real acupuncture, compared with the sham group (+7.5 ± 2.8 vs. −2.9 ± 2.7%, respectively; P < 0.01). By contrast, in naive subjects, arterial diameter did not change during real or sham acupuncture. In both populations, no significant difference was observed between real and sham acupuncture, concerning the time course of blood pressure, radial artery distensibility, and pressure waveform. Our results demonstrate that real acupuncture is associated with an objective vasodilatation of the radial artery in patients regularly exposed to acupuncture, but not in naive subjects.