bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of nitric oxide in β3-adrenoceptor activation on basal tone of internal anal sphincter
Authors: Banwait Kuldip S. , Rattan Satish
Published: March 2015

Effects of activation of β3-adrenoceptor (β3-AR) have not been determined in the spontaneously tonic smooth muscle of the internal anal sphincter (IAS). The effects of disodium (R,R)-5-[2-[2-3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL 316243), a selective β3-AR agonist, on the basal smooth muscle tone and direct release of nitric oxide (NO) by circular smooth muscle strips of the opossum IAS were determined. We also examined the presence of endothelial nitric oxide synthase (eNOS) protein by Western blot studies. CL 316243 produced a concentration-dependent relaxation of the smooth muscle that remained unmodified by different neurohumoral antagonists. The smooth muscle relaxation by CL 316243 was selectively antagonized by L 748337, a β3-AR antagonist. Such relaxation was several times longer than by isoproterenol. The effect of CL 316243 was significantly attenuated by a nonselective NOS inhibitor Nω-nitro-l-arginine (l-NNA) and by putative inhibitor of eNOS l- N5-(1-iminoethyl)-ornithine dihydrochloride (l-NIO). Inhibitors of iNOS [ N-(3-aminomethyl)benzyl acetamide 2HCl] and nNOS {1-[2-(trifluoromethylphenyl)imidazole]} had no effect on this relaxation. Relaxation of the IAS smooth muscle induced by CL 316243 was accompanied by an increased release of NO; this was attenuated by l-NNA and l-NIO. In addition, Western blot studies revealed the presence of eNOS in the circular smooth muscle of the IAS. These data demonstrate potent and protracted IAS smooth muscle relaxation by β3-AR activation, which is partly transduced via NOS, possibly smooth muscle eNOS. Multiple signal-transduction pathways including NOS activation may explain the characteristic IAS relaxation by β3-AR activation. The studies may have therapeutic implications in anorectal motility disorders.
I. Visions of epithelial research
Authors: Montrose Marshall H.
Published: March 2015

Epithelial cells are gatekeepers that sit at the interface between two compartments. By controlling the flow of molecules and information between two compartments, epithelial cells provide unique benefit to the body. This article provides a brief appraisal of our current knowledge about the functions of gastrointestinal epithelial cells as a functionally diverse set of cells mediating transepithelial transport and as a continually renewing layer of cells. The convergence of new methodologies in laser capture microdissection, microarray analyses, microscopic analyses, and generation of mutant animals provides an exciting template for future research.
Tetrahydrobiopterin restores endothelial dysfunction induced by an oral glucose challenge in healthy subjects
Authors: Ihlemann Nikolaj , Rask-Madsen Christian , Perner Anders , Dominguez Helena , Hermann Thomas , Køber Lars , Torp-Pedersen Christian
Published: March 2015

An oral glucose challenge causes transient impairment of endothelial function, probably because of increased oxidative stress. During oxidative stress, endothelial nitric oxide (NO) synthase (eNOS) becomes uncoupled because of decreased bioavailability of tetrahydrobiopterin (BH4), an essential cofactor of eNOS. Therefore, we examined whether an acute supplement of BH4could restore endothelial dysfunction induced by an oral glucose challenge. Healthy subjects were examined in 53 experiments. Forearm blood flow was measured by venous occlusion plethysmography. Dose-response studies were obtained during intra-arterial infusion of serotonin to elicit endothelium-dependent, NO-specific vasodilation and during sodium nitroprusside (SNP) infusion to elicit endothelium-independent vasodilation. Subjects were examined before (fasting) and 1 and 2 h after an oral glucose challenge (75 g) with serotonin ( n = 10) and SNP ( n = 8). On different days (6 R)-5,6,7,8-tetrahydro-l-biopterin dihydrochloride (6R-BH4; n = 10), the active cofactor of eNOS or its stereoisomer (6 S)-5,6,7,8-tetrahydro-l-biopterin sulfate (6S-BH4; n = 10), which is inactive as a cofactor, was added 10 min (500 μg/min) before and during the 1-h postchallenge serotonin dose-response study. In vitro studies showed that 6R-BH4and 6S-BH4were equipotent antioxidants. Serotonin response was reduced by 24 ± 7% (at the highest dose) at 1 h postchallenge compared with fasting ( P = 0.001) and was restored 2 h postchallenge. The reduction was reversed by the administration of 6R-BH4but not by 6S-BH4. SNP responses were slightly increased 1 and 2 h postchallenge (increased by 15 ± 13% at third dose 2 h postchallenge, P = 0.0001). An oral glucose challenge causes transient, NO-specific, endothelial dysfunction, which may be reversed by BH4. Transient postprandial endothelial dysfunction may be partly explained by reduced bioavailability of BH4and NO.
Role of bradykinin in angiotensin-converting enzyme knockout mice
Authors: Xiao Hong D. , Fuchs Sebastien , Cole Justin M. , Disher Kevin M. , Sutliff Roy L. , Bernstein Kenneth E.
Published: March 2015

Angiotensin-converting enzyme (ACE) plays a central role in the renin-angiotensin system. Whereas ACE is responsible for the production of angiotensin II, it is also important in the elimination of bradykinin. Constitutively, the biological function of bradykinin is mediated through the bradykinin B2receptor. ACE knockout mice have a complicated phenotype including very low blood pressure. To investigate the role of bradykinin in the expression of the ACE knockout phenotype, we bred B2receptor knockout mice with ACE knockout mice, thus generating a line of mice deficient in both the B2receptor and ACE. Surprisingly, these mice did not differ from ACE knockout mice in blood pressure, urine concentrating ability, renal pathology, and hematocrit. Thus abnormalities of bradykinin accumulation do not play an important role in the ACE knockout phenotype. Rather, this phenotype appears due to the defective production of angiotensin II.
Unloaded shortening increases peak of Ca2+transients but accelerates their decay in rat single cardiac myocytes
Authors: Yasuda So-ichiro , Sugiura Seiryo , Yamashita Hiroshi , Nishimura Satoshi , Saeki Yasutake , Momomura Shin-ichi , Katoh Kaoru , Nagai Ryozo , Sugi Haruo
Published: March 2015

It is of paramount importance to investigate the relation between the time-dependent change in intracellular Ca2+concentration ([Ca2+]i) (Ca2+transients) and the mechanical activity of isolated single myocytes to understand the regulatory mechanisms of heart function. However, because of technical difficulties in performing mechanical measurements with single myocytes, the simultaneous recording of Ca2+transients and mechanical activity has mainly been performed with multicellular cardiac preparations that give conflicting results concerning Ca2+transients during isometric twitches and during twitches with unloaded shortening. In the present study, we coupled intracellular Ca2+measurement optics with a force measurement system using carbon fibers to examine the relation between Ca2+transients and the mechanical activity of rat single ventricular myocytes over a wide range of load. To minimize the possible load dependence of sarcoplasmic reticulum Ca2+loading, contraction mode was switched at every twitch from unloaded shortening to isometric contraction. During a twitch with unloaded shortening, the Ca2+transients exhibited a higher peak and a higher rate of decay than transients during an isometric twitch. Similarly, when we changed the contraction mode in every pair of twitches, Ca2+transients were dependent only on the mode of contraction. Mechanical uncoupling with 2,3-butanedione monoxime abolished this dependence on the mode of contraction. Our results suggest that Ca2+transients reflect the affinity of troponin C for Ca2+, which is influenced by the change in strain on the thin filament but not by the length change per se.
Changes in excitation-contraction coupling in an isolated ventricular myocyte model of cardiac stunning
Authors: Louch William E. , Ferrier Gregory R. , Howlett Susan E.
Published: March 2015

To investigate cardiac stunning, we recorded intracellular [Ca2+], contractions, and electrical activity in isolated guinea pig ventricular myocytes exposed to simulated ischemia and reperfusion. After equilibration, ischemia was simulated by exposing myocytes to hypoxia, acidosis, hyperkalemia, hypercapnia, lactate accumulation, and substrate deprivation for 30 min at 37°C. Reperfusion was simulated by exposure to Tyrode solution. Field-stimulated myocytes exhibited stunning upon reperfusion. By 10 min of reperfusion, contraction amplitude decreased to 43.0 ± 5.5% of preischemic values ( n = 15, P < 0.05), although action potential configuration and sarcoplasmic reticulum Ca2+stores, assessed with caffeine, were normal. Diastolic [Ca2+] and Ca2+ transients (fura 2) were also normal in stunned myocytes. In voltage-clamped cells, peak L-type Ca2+ current was reduced to 47.4 ± 4.5% of preischemic values at 10 min of reperfusion ( n= 21, P < 0.05). Contractions elicited by Ca2+-induced Ca2+ release and the voltage-sensitive release mechanism were both depressed in reperfusion. Our observations suggest that stunning is associated with reduced L-type Ca2+ current but that alterations in Ca2+ homeostasis and release are not directly responsible for stunning.
PYK2 expression and phosphorylation increases in pressure overload-induced left ventricular hypertrophy
Authors: Bayer Allison L. , Heidkamp Maria C. , Patel Nehu , Porter Michael J. , Engman Steven J. , Samarel Allen M.
Published: March 2015

Proline-rich tyrosine kinase 2 (PYK2) is a member of the focal adhesion kinase (FAK) family of nonreceptor protein tyrosine kinases. PYK2 has been implicated in linking G protein-coupled receptors to activation of mitogen-activated protein kinase cascades and cellular growth in a variety of cell types. To determine whether PYK2 expression and phosphorylation is altered in left ventricular (LV) myocardium undergoing LV hypertrophy (LVH) and heart failure in vivo, suprarenal abdominal aortic coarctation was performed in 160-g male Sprague-Dawley rats. Immunohistochemistry and Western blotting were performed on LV tissue 1, 8, and 24 wk after aortic banding. Aortic banding produced sustained hypertension and gradually developing LVH. PYK2 levels were increased 1.8 ± 0.2-, 2.7 ± 0.6-, and 2.0 ± 0.2-fold in 1-, 8-, and 24-wk banded animals compared with their respective sham-operated controls. The increase in PYK2 expression was paralleled by an increase in PYK2 phosphorylation, both of which preceded the development of LVH. Immunohistochemistry revealed that enhanced PYK2 expression occurred predominantly in the cardiomyocyte population. Furthermore, there was a high degree of correlation ( R = 0.75; P< 0.001) between the level of PYK2 and the degree of LVH in 24-wk sham and banded animals. In contrast, FAK levels and FAK phosphorylation were not increased before the development of LVH. However, there was a high degree of correlation (R = 0.68; P < 0.001) between the level of FAK and the degree of LVH in 24-wk sham and banded rats. There was also a significant increase in the ratio of phosphospecific anti-FAK to FAK at this time point. These data are consistent with a role for PYK2 in the induction of pressure overload-induced cardiomyocyte hypertrophy, and suggest that PYK2 and FAK have distinctly different roles in LVH progression.
Cerebrovascular autoregulation is profoundly impaired in mice overexpressing amyloid precursor protein
Authors: Niwa Kiyoshi , Kazama Ken , Younkin Linda , Younkin Steven G. , Carlson George A. , Iadecola Costantino
Published: March 2015

The amyloid-β (Aβ) peptide, which is derived from the amyloid precursor protein (APP), is involved in the pathogenesis of Alzheimer's dementia and impairs endothelium-dependent vasodilation in cerebral vessels. We investigated whether cerebrovascular autoregulation, i.e., the ability of the cerebral circulation to maintain flow in the face of changes in mean arterial pressure (MAP), is impaired in transgenic mice that overexpress APP and Aβ. Neocortical cerebral blood flow (CBF) was monitored by laser-Doppler flowmetry in anesthetized APP(+) and APP(−) mice. MAP was elevated by intravenous infusion of phenylephrine and reduced by controlled exsanguination. In APP(−) mice, autoregulation was preserved. However, in APP(+) mice, autoregulation was markedly disrupted. The magnitude of the disruption was linearly related to brain Aβ concentration. The failure of autoregulation was paralleled by impairment of the CBF response to endothelium-dependent vasodilators. Thus Aβ disrupts a critical homeostatic mechanism of the cerebral circulation and renders CBF highly dependent on MAP. The resulting alterations in cerebral perfusion may play a role in the brain dysfunction and periventricular white-matter changes associated with Alzheimer's dementia.
Myocardial function defined by strain rate and strain during alterations in inotropic states and heart rate
Authors: Weidemann Frank , Jamal Fadi , Sutherland George R. , Claus Piet , Kowalski Miroslaw , Hatle Liv , De Scheerder Ivan , Bijnens Bart , Rademakers Frank E.
Published: March 2015

For porcine myocardium, ultrasonic regional deformation parameters, systolic strain (εsys) and peak systolic strain rate (SRsys), were compared with stroke volume (SV) and contractility [contractility index (CI)] measured as the ratio of end-systolic strain to end-systolic wall stress. Heart rate (HR) and contractility were varied by atrial pacing (AP = 120–180 beats/min, n = 7), incremental dobutamine infusion (DI = 2.5–20 μg · kg−1 · min−1, n = 7), or continuous esmolol infusion (0.5 mg · kg−1 · min−1) + subsequent pacing (120–180 beats/min) (EI group, n= 6). Baseline SRsys and εsys averaged 5.0 ± 0.4 s−1 and 60 ± 4%. SRsysand CI increased linearly with DI (20 μg · kg−1 · min−1; SRsys = 9.9 ± 0.7 s−1, P < 0.0001) and decreased with EI (SRsys = 3.4 ± 0.1 s−1, P < 0.01). During pacing, SRsys and CI remained unchanged in the AP and EI groups. During DI, εsys and SV initially increased (5 μg · kg−1 · min−1; εsys = 77 ± 6%, P < 0.01) and then progressively returned to baseline. During EI, SV and εsys decreased (εsys = 38 ± 2%, P < 0.001). Pacing also decreased SV and εsys in the AP (180 beats/min; εsys = 36 ± 2%, P < 0.001) and EI groups (180 beats/min; εsys = 25 ± 3%, P< 0.001). Thus, for normal myocardium, SRsys reflects regional contractile function (being relatively independent of HR), whereas εsys reflects changes in SV.
CaMKII-dependent reactivation of SR Ca2+uptake and contractile recovery during intracellular acidosis
Authors: Nomura Noriyuki , Satoh Hiroshi , Terada Hajime , Matsunaga Masaki , Watanabe Hiroshi , Hayashi Hideharu
Published: March 2015

In hearts, intracellular acidosis disturbs contractile performance by decreasing myofibrillar Ca2+response, but contraction recovers at prolonged acidosis. We examined the mechanism and physiological implication of the contractile recovery during acidosis in rat ventricular myocytes. During the initial 4 min of acidosis, the twitch cell shortening decreased from 2.3 ± 0.3% of diastolic length to 0.2 ± 0.1% (means ± SE, P < 0.05, n = 14), but in nine of these cells, contractile function spontaneously recovered to 1.5 ± 0.3% at 10 min ( P < 0.05 vs. that at 4 min). During the depression phase, both the diastolic intracellular Ca2+concentration ([Ca2+]i) and Ca2+transient (CaT) amplitude increased, and the twitch [Ca2+]idecline prolonged significantly ( P < 0.05). In the cells that recovered, a further increase in CaT amplitude and a reacceleration of twitch [Ca2+]idecline were observed. The increase in diastolic [Ca2+]iwas less extensive than the increase in the cells that did not recover ( n = 5). Blockade of sarcoplasmic reticulum (SR) function by ryanodine (10 μM) and thapsigargin (1 μM) or a selective inhibitor of Ca2+-calmodulin kinase II, 2-[ N- (2-hydroxyethyl)- N-(4-methoxybenzenesulfonyl)] amino- N-(4-chlorocinnamyl)- N-methyl benzylamine (1 μM) completely abolished the reacceleration of twitch [Ca2+]idecline and almost eliminated the contractile recovery. We concluded that during prolonged acidosis, Ca2+-calmodulin kinase II-dependent reactivation of SR Ca2+uptake could increase SR Ca2+content and CaT amplitude. This recovery can compensate for the decreased myofibrillar Ca2+response, but may also cause Ca2+overload after returning to physiological pHi.
Enalapril attenuates endothelin-1-induced hypertension via increased kinin survival
Authors: Elmarakby Ahmed A. , Morsing Peter , Pollock David M.
Published: March 2015

Recent studies have shown that angiotensin-converting enzyme (ACE) inhibitors attenuate endothelin-1 (ET-1)-induced hypertension, but the mechanisms for this effect have not been clarified. Initial experiments were conducted to contrast the effect of the ACE inhibitor enalapril, the combined ACE-neutral endopeptidase inhibitor omapatrilat, and the angiotensin II receptor antagonist candesartan on the hypertensive and renal response to ET-1 in anesthetized Sprague-Dawley rats. Acute intravenous infusion of ET-1 (10 pmol · kg−1 · min−1) for 60 min significantly increased mean arterial pressure (MAP) from 125 ± 8 to 145 ± 8 mmHg ( P < 0.05) and significantly decreased glomerular filtration rate (GFR) from 0.31 ± 0.09 to 0.13 ± 0.05 ml · min−1 · 100 g kidney wt−1. Pretreatment with enalapril (10 mg/kg iv) before ET-1 infusion inhibited the increase in MAP (121 ± 4 vs. 126 ± 4 mmHg) before and during ET-1 infusion, respectively ( P < 0.05) without blocking the effect of ET-1 on GFR. In contrast, neither omapatrilat (30 mg/kg) nor candesartan (10 mg/kg) had any effect on ET-1-induced increases in MAP or decreases in GFR. To determine whether the effect of enalapril was due to the decrease in angiotensin II or increase in kinin formation, rats were given REF-000359 (1 mg/kg iv), a selective B2 receptor antagonist, with or without enalapril before ET-1 infusion. REF-000359 completely blocked the effect of enalapril on ET-1 infusion (MAP was 117 ± 5 vs. 135 ± 5 mmHg before and during ET-1 infusion, respectively, P < 0.05). REF-000359 alone had no effect on the response to ET-1 infusion (MAP was 117 ± 4 vs. 144 ± 4 mmHg before and during ET-1 infusion, respectively, P < 0.05). REF-000359 with or without enalapril had no significant effect on the ability of ET-1 infusion to decrease GFR. These findings support the hypothesis that decreased catabolism of bradykinin and its subsequent vasodilator activity oppose the actions of ET-1 to increase MAP.
Bioenergetic remodeling of heart during treatment of spontaneously hypertensive rats with enalapril
Authors: Leary S. C. , Michaud D. , Lyons C. N. , Hale T. M. , Bushfield T. L. , Adams M. A. , Moyes C. D.
Published: March 2015

We used spontaneously hypertensive rats to study remodeling of cardiac bioenergetics associated with changes in blood pressure. Blood pressure was manipulated with aggressive antihypertensive treatment combining low dietary salt and the angiotensin-converting enzyme inhibitor enalapril. Successive cycles of 2 wk on, 2 wk off treatment led to rapid, reversible changes in left ventricular (LV) mass (30% change in <10 days). Despite changes in LV mass, specific activities of bioenergetic (cytochrome- c oxidase, citrate synthase, lactate dehydrogenase) and reactive oxygen species (ROS) (total cellular superoxide dismutase) enzymes were actively maintained within relatively narrow ranges regardless of treatment duration, organismal age, or transmural region. Although enalapril led to parallel declines in mitochondrial enzyme content and ventricular mass, total ventricular mtDNA content was unaffected. Altered enzymatic content occurred without significant changes in relevant mRNA and protein levels. Transcript levels of gene products involved in mtDNA maintenance (Tfam), mitochondrial protein degradation (LON protease), fusion (fuzzy onion homolog), and fission (dynamin-like protein, synaptojanin-2α) were also unchanged. In contrast, enalapril-mediated ventricular and mitochondrial remodeling was accompanied by a twofold increase in specific activity of catalase, an indicator of oxidative stress, suggesting that rapid cardiac adaptation is accompanied by tight regulation of mitochondrial enzyme activities and increased ROS production.
Downregulation of bradykinin B2receptor in human fibroblasts during prolonged agonist exposure
Authors: Blaukat Andree , Micke Patrick , Kalatskaya Irina , Faussner Alexander , Müller-Esterl Werner
Published: March 2015

Sustained activation of G protein-coupled receptors results in an attenuation of cellular responses, a phenomenon termed desensitization. Whereas mechanisms for rapid desensitization of ligand-receptor-G protein-effector systems are relatively well characterized, much less is known about long-term adaptation processes that occur in the continuous presence of an agonist. Here we have studied the fate of endogenously expressed bradykinin B2receptors on human fibroblasts during prolonged agonist treatment. Stimulation with bradykinin for up to 24 h resulted in a 50% reduction of surface binding sites that was paralleled by a similar decrease of total B2receptor protein followed by Western blotting using monoclonal antibodies to the B2receptor. Whereas B2receptor mRNA levels did not change during 24 h of agonist treatment, B2receptor de novo synthesis was attenuated by 35–50%, indicating translational control of B2receptor levels. Furthermore, the half-life of B2receptor protein was shortened by 20–40% as shown by35S-labeled pulse-chase and immunoprecipitation experiments. This study demonstrates that bradykinin B2receptor expression during long-term agonist treatment is primarily regulated on the (post)translational level, i.e., by attenuation of de novo synthesis and by reduction of receptor stability.
Arterial intima-media thickness: site-specific associations with HRT and habitual exercise
Authors: Moreau Kerrie L. , Donato Anthony J. , Seals Douglas R. , Dinenno Frank A. , Blackett Sharon D. , Hoetzer Greta L. , Desouza Christopher A. , Tanaka Hirofumi
Published: March 2015

We determined the site-specific relations of hormone replacement therapy (HRT) and habitual exercise status with intima-media thickness (IMT) in both elastic (carotid) and muscular (femoral) arteries in 77 healthy postmenopausal women: 43 women were sedentary (20 no-HRT and 23 HRT users) and 34 women were endurance trained (14 no-HRT and 20 HRT users). Femoral IMT was not different among the sedentary HRT and endurance-trained no-HRT and HRT groups, but was lower ( P < 0.005) in these three groups than in the sedentary no-HRT women. There were no significant group differences in carotid IMT. However, in older women (≥65 yrs) carotid IMT was smaller ( P < 0.05) in HRT compared with no-HRT women. We conclude that both endurance training and HRT status are independently associated with a smaller IMT and these effects are evident primarily in muscular arteries. These results suggest that HRT and habitual exercise may protect postmenopausal women against cardiovascular disease through influences on IMT. The site-specific relations may be due to a greater number of smooth muscle cells and plasticity of muscular arteries compared with elastic arteries and/or differences in heterogeneous influences such as metabolic requirements and hydrostatic pressures.
Kininase I-type carboxypeptidases enhance nitric oxide production in endothelial cells by generating bradykinin B1receptor agonists
Authors: Sangsree Sakonwun , Brovkovych Viktor , Minshall Richard D. , Skidgel Randal A.
Published: March 2015

Kininase I-type carboxypeptidases convert native kinin agonists for B2receptors into B1receptor agonists by specifically removing the COOH-terminal Arg residue. The membrane localization of carboxypeptidase M (CPM) and carboxypeptidase D (CPD) make them ideally situated to regulate kinin activity. Nitric oxide (NO) release from human lung microvascular endothelial cells (HLMVEC) was measured directly in real time with a porphyrinic microsensor. Bradykinin (1–100 nM) elicited a transient (5 min) peak of generation of NO that was blocked by the B2antagonist HOE 140, whereas B1agonist des-Arg10-kallidin caused a small linear increase in NO over 20 min. Treatment of HLMVEC with 5 ng/ml interleukin-1β and 200 U/ml interferon-γ for 16 h upregulated B1receptors as shown by an approximately fourfold increase in prolonged (>20 min) output of NO in response to des-Arg10-kallidin, which was blocked by the B1antagonist des-Arg10-Leu9-kallidin. B2receptor agonists bradykinin or kallidin also generated prolonged NO production in treated HLMVEC, which was significantly reduced by either a B1antagonist or carboxypeptidase inhibitor, and completely abolished with a combination of B1and B2receptor antagonists. Furthermore, CPM and CPD activities were increased about twofold in membrane fractions of HLMVEC treated with interleukin-1β and interferon-γ compared with control cells. Immunostaining localized CPD primarily in a perinuclear/Golgi region, whereas CPM was on the cell membrane. These data show that cellular kininase I-type carboxypeptidases can enhance kinin signaling and NO production by converting B2agonists to B1agonists, especially in inflammatory conditions.
Effects of oxygen tension on energetics of cultured vascular smooth muscle
Authors: Lindqvist Anders , Dreja Karl , Swärd Karl , Hellstrand Per
Published: March 2015

Chronic hypoxia is a clinically important condition known to cause vascular abnormalities. To investigate the cellular mechanisms involved, we kept rings of a rat tail artery for 4 days in hypoxic culture (HC) or normoxic culture (NC) (Po 2 = 14 vs. 110 mmHg) and then measured contractility, oxygen consumption ( J o2 ), and lactate production ( J lac) in oxygenated medium. Compared with fresh rings, basal ATP turnover ( J ATP) was decreased in HC, but not in NC, with a shift from oxidative toward glycolytic metabolism. J o2 during mitochondrial uncoupling was reduced by HC but not by NC. Glycogen stores were increased 40-fold by HC and fourfold by NC. Maximum tension in response to norepinephrine and the Jo2 versus tension relationship ( J o2 vs. high K+ elicited force) were unaffected by either HC or NC. Force transients in response to caffeine were increased in HC, whereas intracellular Ca2+ wave activity during adrenergic stimulation was decreased. Protein synthesis rate was reduced by HC. The results show that long-term hypoxia depresses basal energy turnover, impairs mitochondrial capacity, and alters Ca2+homeostasis, but does not affect contractile energetics. These alterations may form a basis for vascular damage by chronic hypoxia.
Balanced expression of mitochondrial apoptosis regulatory proteins correlates with long-term survival of cardiac allografts
Authors: Tung Thomas C. , Cui Guanggen , Oshima Kiyohiro , Laks Hillel , Sen Luyi
Published: March 2015

Abnormal regulation of apoptosis is observed in ischemic injury and may contribute to the pathogenesis of atherosclerosis. However, its role in cardiac allograft vasculopathy (CAV), the fundamental lesion of chronic rejection (CR) in heart transplantation, remains uncertain. To clarify this issue, apoptosis was quantitated in myocardium and coronary arteries from 5 cardiac allograft donors (NL) and explanted hearts of 24 patients with ischemic cardiomyopathy (IsCM) and 15 patients with CR. Tissue samples were analyzed via end-labeling fragmented DNA [via deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)] and immunoblotting for activated caspase-3 and -9. Myocyte apoptosis assessed by TUNEL was similarly increased over NL (0.21%) in both the CR (0.88%; P < 0.01) and IsCM (0.88%; P < 0.01) groups. Activated caspase-9 levels were significantly higher in CR (14.7%) compared with IsCM (6.9%; P < 0.01) and NL (0%) groups, whereas activated caspase-3 levels were similarly elevated in both CR and IsCM (7.8 and 6.5% vs. 0% in NL; P < 0.01 and P < 0.05) groups. Expression of myocardial Bcl-2 and Bax was increased in CR compared with both NL (Bax, 4.3-fold; P < 0.01; Bcl-2, 5.9-fold; P < 0.01) and IsCM (IsCM: Bax, 2.2-fold; P < 0.05; Bcl-2, 3.2-fold; P < 0.01) groups. The rate of apoptosis and the Bcl-2/Bax ratio independently correlated to graft survival in CR (activation of caspase-9: r = 0.87; P < 0.01; Bcl-2/Bax: r = 0.57; P = 0.05). Compared with native atherosclerosis, coronary arteries with CAV showed more medial apoptosis (7.8-fold; P < 0.01) and higher Bcl-2 levels (5.1-fold; P < 0.01) with lower Bax levels (threefold; P < 0.05) in the intima. These results indicate that abnormal Bcl-2 and Bax expression in myocardium and coronary arteries of cardiac allografts with CR is distinct from that in IsCM and suggest that balancing Bcl-2 to Bax in transplanted hearts promotes long-term graft survival.
Differential PDGF secretion by graft and aortic SMC in response to oxidized LDL
Authors: Absood Afaf , Furutani Akira , Kawamura Tsutomu , Graham Linda M.
Published: March 2015

Smooth muscle cells (SMC) from prosthetic vascular grafts constitutively secrete higher levels of platelet-derived growth factor-AA (PDGF-AA) than aortic SMC. Lipid oxidation products accumulate in grafts and may stimulate PDGF production. The effect of oxidized low-density lipoprotein (oxLDL) on PDGF-AA secretion by aortic and graft SMC was compared. SMC isolated from canine thoracic aorta or Dacron thoracoabdominal grafts ( n = 10) were incubated with native LDL or oxLDL (0–400 μg/ml) for 72 h. PDGF-AA in the conditioned medium was measured with enzyme-linked immunosorbent assay. OxLDL increased PDGF-AA production by graft SMC from 78 ± 2 to 256 ± 16 pg PDGF/μg DNA and aortic SMC from 21 ± 1 to 40 ± 2 pg PDGF/μg DNA. Native LDL had no effect. N-acetylcysteine inhibited oxLDL-induced PDGF increase. Both superoxide and H2O2 stimulated PDGF secretion by graft SMC had little effect on aortic SMC. Our results suggest that PDGF production by graft (synthetic) SMC is more sensitive to stimulation by oxidative stress than aortic (contractile) SMC. Lipid oxidation products that accumulate in prosthetic vascular grafts can cause an oxidative stress, which stimulates PDGF production by graft SMC. PDGF can induce migration of aortic SMC onto the graft, contributing to the development of intimal hyperplasia.
Dynamic cerebral autoregulation remains stable during physical challenge in healthy persons
Authors: Brys Miroslaw , Brown Clive M. , Marthol Harald , Franta Renate , Hilz Max J.
Published: March 2015

The effects of physical activity on cerebral blood flow (CBF) and cerebral autoregulation (CA) have not yet been fully evaluated. There is controversy as to whether increasing heart rate (HR), blood pressure (BP), and sympathetic and metabolic activity with altered levels of CO2 might compromise CBF and CA. To evaluate these effects, we studied middle cerebral artery blood flow velocity (CBFV) and CA in 40 healthy young adults at rest and during increasing levels of physical exercise. We continuously monitored HR, BP, end-expiratory CO2, and CBFV with transcranial Doppler sonography at rest and during stepwise ergometric challenge at 50, 100, and 150 W. The modulation of BP and CBFV in the low-frequency (LF) range (0.04–0.14 Hz) was calculated with an autoregression algorithm. CA was evaluated by calculating the phase shift angle and gain between BP and CBFV oscillations in the LF range. The LF BP-CBFV gain was then normalized by conductance. Cerebrovascular resistance (CVR) was calculated as mean BP adjusted to brain level divided by mean CBFV. HR, BP, CO2, and CBFV increased significantly with exercise. Phase shift angle, absolute and normalized LF BP-CBFV gain, and CVR, however, remained stable. Stable phase shift, LF BP-CBFV gain, and CVR demonstrate that progressive physical exercise does not alter CA despite increasing HR, BP, and CO2. CA seems to compensate for the hemodynamic effects and increasing CO2 levels during exercise.
Cerebrovascular response to decreased hematocrit: effect of cell-free hemoglobin, plasma viscosity, and CO2
Authors: Rebel Annette , Ulatowski John A. , Kwansa Herman , Bucci Enrico , Koehler Raymond C.
Published: March 2015

The effect of transfusing a nonextravasating, zero-link polymer of cell-free hemoglobin on pial arteriolar diameter, cerebral blood flow (CBF), and O2 transport (CBF × arterial O2 content) was compared with that of transfusing an albumin solution at equivalent reductions in hematocrit (∼19%) in anesthetized cats. The influence of viscosity was assessed by coinfusion of a high-viscosity solution of polyvinylpyrrolidone (PVP), which increased plasma viscosity two- to threefold. Exchange transfusion of a 5% albumin solution resulted in pial arteriolar dilation, increased CBF, and unchanged O2 transport, whereas there were no significant changes over time in a control group. Exchange transfusion of a 12% polymeric hemoglobin solution resulted in pial arteriolar constriction and unchanged CBF and O2 transport. Coinfusion of PVP with albumin produced pial arteriolar dilation that was similar to that obtained with transfusion of albumin alone. In contrast, coinfusion of PVP with hemoglobin converted the constrictor response to a dilator response that prevented a decrease in CBF. Pial arteriolar dilation to hypercapnia was unimpaired in groups transfused with albumin or hemoglobin alone but was attenuated in the largest vessels in albumin and hemoglobin groups coinfused with PVP. Unexpectedly, hypocapnic vasoconstriction was blunted in all groups after transfusion of albumin or hemoglobin alone or with PVP. We conclude that 1) the increase in arteriolar diameter after albumin transfusion represents a compensatory response that prevents decreased O2 transport at reduced O2-carrying capacity, 2) the decrease in diameter associated with near-normal O2-carrying capacity after cell-free polymeric hemoglobin transfusion represents a compensatory mechanism that prevents increased O2 transport at reduced blood viscosity, 3) pial arterioles are capable of dilating to an increase in plasma viscosity when hemoglobin is present in the plasma, 4) decreasing hematocrit does not impair pial arteriolar dilation to hypercapnia unless plasma viscosity is increased, and 5) pial arteriolar constriction to hypocapnia is impaired at reduced hematocrit independently of O2-carrying capacity.
Superoxide contributes to vascular dysfunction in mice that express human renin and angiotensinogen
Authors: Didion Sean P. , Ryan Michael J. , Baumbach Gary L. , Sigmund Curt D. , Faraci Frank M.
Published: March 2015

This study examined vascular function and the role of superoxide in mice that chronically express human renin (R+) and human angiotensinogen (A+). Responses of aortas from R+/A+ mice and from their normotensive littermates (RA− mice) were examined in vitro. Endothelium-dependent relaxation to acetylcholine was impaired in vessels from R+/A+ mice (e.g., maximal relaxation to 100 μM acetylcholine was 45 ± 5% and 65 ± 3% in R+/A+ and RA− mice, respectively; P < 0.05). Relaxation was also impaired to the endothelium-independent dilators authentic nitric oxide and nitroprusside in vessels from R+/A+ mice. Maximal vasorelaxation to the endothelium-independent, non-nitric oxide dilator papaverine was similar in R+/A+ and RA− mice. Incubation of vessels from R+/A+ mice with Tiron (1 mM), a superoxide scavenger, improved relaxation to acetylcholine, nitric oxide, and nitroprusside. In contrast, incubation with diethyldithiocarbamate (1 mM), an inhibitor of copper-containing SODs, reduced acetylcholine- and nitroprusside-induced relaxation in vessels from both R+/A+ and RA− mice. Basal superoxide levels, measured with lucigenin-enhanced chemiluminescence (5 μM lucigenin) and hydroethidine-based fluorescent confocal microscopy, were higher in vessels from R+/A+ mice and were Tiron and polyethylene glycol-SOD sensitive. These results suggest that increased superoxide contributes to impaired nitric oxide-mediated relaxation in this genetic model of chronic angiotensin II-dependent hypertension.
Differential involvement of MMP-2 and VEGF during muscle stretch- versus shear stress-induced angiogenesis
Authors: Rivilis Irina , Milkiewicz Malgorzata , Boyd Pamela , Goldstein Jason , Brown Margaret D. , Egginton Stuart , Hansen Fay M. , Hudlicka Olga , Haas Tara L.
Published: March 2015

Capillary growth in skeletal muscle occurs via the dissimilar processes of abluminal sprouting or longitudinal splitting, which can be initiated by muscle stretch and elevated shear stress, respectively. The distinct morphological hallmarks of these types of capillary growth suggest that discrete sets of angiogenic mediators play a role in each situation. Because proteolysis and proliferation are two key steps associated with capillary growth, we tested whether differences in the regulation of matrix metalloproteinases (MMPs) or VEGF may be associated with the two types of capillary growth. We found significant increases in MMP-2 total protein and percent activation, and membrane type-1 MMP mRNA levels, compared with controls after muscle stretch but not after shear stress stimulation. In contrast, VEGF protein and endothelial cell proliferation increased after either angiogenic stimulus. We observed that MMP-2 regulation occurs independent of VEGF signaling, because VEGF did not induce MMP-2 production or activation in isolated endothelial cells. Our data suggest that the involvement of MMPs in capillary growth is dependent on the nature of the angiogenic stimulus.
Hemodynamic and renal effects of low-dose brain natriuretic peptide infusion in humans: a randomized, placebo-controlled crossover study
Authors: van der Zander Kim , Houben Alphons J. H. M. , Hofstra Leo , Kroon Abraham A. , de Leeuw Peter W.
Published: March 2015

Brain natriuretic peptide (BNP) is a cardiac hormone with natriuretic activity. The aim of this study was to investigate the cardiovascular effects of pathophysiological levels of BNP on central hemodynamics, cardiac function, renal hemodynamics and function, and microvascular hemodynamics in healthy subjects. In this double-blind, placebo-controlled crossover study, we intravenously infused BNP (4 pmol · kg–1 · min–1) or placebo for 1 h on two separate days in 12 healthy subjects (mean age, 60 ± 5 yr). Nailfold and conjunctival capillary density, finger-skin (thermoregulatory) microvascular blood flow, and cardiac output were studied before and after infusion using intravital videomicroscopy, laser-Doppler fluxmetry, and echocardiography, respectively. Furthermore, during infusion, we measured the effective renal plasma flow and glomerular filtration rate using p-aminohippurate and inulin clearances. Blood pressure and heart rate were monitored for all measurements. Compared with placebo, BNP significantly decreased stroke volume with a tendency to decrease cardiac output. With subjects in the sitting position, mean arterial pressure decreased and heart rate increased after BNP infusion, whereas with subjects in the supine position, these variables remained unchanged. BNP increased natriuresis, diuresis, glomerular filtration rate, filtration fraction, and filtered load of Na+ compared with placebo, whereas effective renal plasma flow did not change. BNP did not affect the microvascular capillary density of conjunctiva and skin, microvascular blood flow, total skin oxygen capacity, and postocclusive recruitment. These results suggest that BNP has predominantly central and renal hemodynamic effects; however, it does not influence peripheral microcirculation in skin and conjunctiva.
3-D MRI assessment of regional left ventricular systolic wall stress in patients with reperfused MI
Authors: Delépine Stephane , Furber Alain P. , Beygui Farzin , Prunier Fabrice , Balzer Philippe , Le Jeune Jean-Jacques , Geslin Philippe
Published: March 2015

The goal of this study was to assess the regional variations of end-systolic wall stress in patients with reperfused Q wave acute myocardial infarction (AMI), with the use of a three-dimensional (3-D) approach. Fifteen normal volunteers and fifty patients with reperfused AMI underwent cardiac MRI that used a short-axis fast-gradient-echo sequence. The end-systolic wall stress was calculated with the use of the Grossman formula with the radius and the wall thickness defined with a 3-D approach using the tridimensional curvature. The mean wall stress was significantly increased at each level of the short-axis plane only in patients with anterior AMI. When calculated at a regional level in patients with anterior AMI, wall stress significantly increased in anterior sector as well as normal sector. In patients with inferior AMI, wall stress significantly increased only in inferior and lateral sectors. In conclusion, the quantification of regional wall stress by cardiac MRI is better with the 3D approach than other methods for precise evaluation in patients with AMI. Despite early reperfusion, the wall stress remained high in patients with anterior AMI.
Role of cardiac eNOS expression during pregnancy in the coupling of myocardial oxygen consumption to cardiac work
Authors: Linke Axel , Li Wei , Huang Harer , Wang Ziping , Hintze Thomas H.
Published: March 2015

We assessed whether pregnancy results in enhanced nitric oxide (NO)-mediated control of myocardial oxygen consumption. Rats were studied before (C), at 1 wk (1w) or 2 wk (2w) of pregnancy, and at 4 days after giving birth (−4d). Left ventricular endothelial NO synthase (eNOS) protein expression was determined by immunoblotting. Oxygen consumption of left ventricular tissue samples was measured in vitro in response to increasing doses of bradykinin with or without addition of the NOS inhibitor N G-nitro-l-arginine methyl ester (l-NAME). Echocardiography indicated an increased cardiac output during pregnancy. Myocardial eNOS protein expression significantly increased by 46 ± 9 and 39 ± 8% at 1w and 2w, respectively, and returned to control levels at −4d. Bradykinin (10−4 M) decreased cardiac oxygen consumption in a NO-dependent manner by 17 ± 2% at C, by 21 ± 2% at 1w, by 24 ± 2% at 2w ( P < 0.05 vs. C and −4d), and by 18 ± 1% at −4d. Myocardial eNOS protein expression is transiently increased during pregnancy in rats, and this increase is associated with enhanced NO-dependent control of myocardial oxygen consumption at a time when cardiac output is increased.
Shear properties of passive ventricular myocardium
Authors: Dokos Socrates , Smaill Bruce H. , Young Alistair A. , LeGrice Ian J.
Published: March 2015

We examined the shear properties of passive ventricular myocardium in six pig hearts. Samples (3 × 3 × 3 mm) were cut from adjacent regions of the lateral left ventricular midwall, with sides aligned with the principal material axes. Four cycles of sinusoidal simple shear (maximum shear displacements of 0.1–0.5) were applied separately to each specimen in two orthogonal directions. Resulting forces along the three axes were measured. Three specimens from each heart were tested in different orientations to cover all six modes of simple shear deformation. Passive myocardium has nonlinear viscoelastic shear properties with reproducible, directionally dependent softening as strain is increased. Shear properties were clearly anisotropic with respect to the three principal material directions: passive ventricular myocardium is least resistant to simple shear displacements imposed in the plane of the myocardial layers and most resistant to shear deformations that produce extension of the myocyte axis. Comparison of results for the six different shear modes suggests that simple shear deformation is resisted by elastic elements aligned with the microstructural axes of the tissue.
Cardiac hypertrophy and altered β-adrenergic signaling in transgenic mice that express the amino terminus of β-ARK1
Authors: Keys Janelle R. , Greene Emily A. , Cooper Chris J. , Naga Prasad Sathyamangla V. , Rockman Howard A. , Koch Walter J.
Published: March 2015

The G protein-coupled receptor (GPCR) kinase β-adrenergic receptor (β-AR) kinase-1 (β-ARK1) is elevated during heart failure; however, its role is not fully understood. β-ARK1 contains several domains that are capable of protein-protein interactions that may play critical roles in the regulation of GPCR signaling. In this study, we developed a novel line of transgenic mice that express an amino-terminal peptide of β-ARK1 that is comprised of amino acid residues 50–145 (β-ARKnt) in the heart to determine whether this domain has any functional significance in vivo. Surprisingly, the β-ARKnt transgenic mice presented with cardiac hypertrophy. Our data suggest that the phenotype was driven via an enhanced β-AR system, as β-ARKnt mice had elevated cardiac β-AR density. Moreover, administration of a β-AR antagonist reversed hypertrophy in these mice. Interestingly, signaling through the β-AR in response to agonist stimulation was not enhanced in these mice. Thus the amino terminus of β-ARK1 appears to be critical for normal β-AR regulation in vivo, which further supports the hypothesis that β-ARK1 plays a key role in normal and compromised cardiac GPCR signaling.
Adenylate kinase AK1 knockout heart: energetics and functional performance under ischemia-reperfusion
Authors: Pucar Darko , Bast Peter , Gumina Richard J. , Lim Lynette , Drahl Carmen , Juranic Nenad , Macura Slobodan , Janssen Edwin , Wieringa Be , Terzic Andre , Dzeja Petras P.
Published: March 2015

Deletion of the major adenylate kinase AK1 isoform, which catalyzes adenine nucleotide exchange, disrupts cellular energetic economy and compromises metabolic signal transduction. However, the consequences of deleting the AK1 gene on cardiac energetic dynamics and performance in the setting of ischemia-reperfusion have not been determined. Here, at the onset of ischemia, AK1 knockout mice hearts displayed accelerated loss of contractile force compared with wild-type controls, indicating reduced tolerance to ischemic stress. On reperfusion, AK1 knockout hearts demonstrated reduced nucleotide salvage, resulting in lower ATP, GTP, ADP, and GDP levels and an altered metabolic steady state associated with diminished ATP-to-Piand creatine phosphate-to-Piratios. Postischemic AK1 knockout hearts maintained ∼40% of β-phosphoryl turnover, suggesting increased phosphotransfer flux through remaining adenylate kinase isoforms. This was associated with sustained creatine kinase flux and elevated cellular glucose-6-phosphate levels as the cellular energetic system adapted to deletion of AK1. Such metabolic rearrangements, along with sustained ATP-to-ADP ratio and total ATP turnover rate, maintained postischemic contractile recovery of AK1 knockout hearts at wild-type levels. Thus deletion of the AK1 gene reveals that adenylate kinase phosphotransfer supports myocardial function on initiation of ischemic stress and safeguards intracellular nucleotide pools in postischemic recovery.