bluuebunny/crossref_metadata_2025 huggingface dataset:
Infarction alters both the distribution and noradrenergic properties of cardiac sympathetic neurons
Authors: Li Wei , Knowlton David , Van Winkle Donna M. , Habecker Beth A.
Published: May 2004

Regional changes occur in the sympathetic innervation of the heart after myocardial infarction (MI), including loss of norepinephrine (NE) uptake and depletion of neuronal NE. This apparent denervation is accompanied by increased cardiac NE spillover. One potential explanation for these apparently contradictory findings is that the sympathetic neurons innervating the heart are exposed to environmental stimuli that alter neuronal function. To understand the changes that occur in the innervation of the heart after MI, immunohistochemical, biochemical, and molecular analyses were carried out in the heart and stellate ganglia of control and MI rats. Immunohistochemistry with panneuronal markers revealed extensive denervation in the left ventricle (LV) below the infarct, but sympathetic nerve fibers were retained in the base of the heart. Western blot analysis revealed that tyrosine hydroxylase (TH) expression (normalized to a panneuronal marker) was increased significantly in the base of the heart and in the stellate ganglia but decreased in the LV below the MI. NE transporter (NET) binding sites, normalized to total protein, were unchanged, except in the LV, where [3H]nisoxetine binding was decreased. TH mRNA was increased significantly in the left and right stellate ganglia after MI, while NET mRNA was not. In the base of the heart, increased TH coupled with no change in NET may explain the increase in extracellular NE observed after MI. Coupled with substantial denervation in the LV, these changes likely contribute to the onset of cardiac arrhythmias.
Glutathione S-transferase overexpression protects against anthracycline-induced H9C2 cell death
Authors: L'Ecuyer Thomas , Allebban Zuhair , Thomas Ronald , Vander Heide Richard
Published: May 2004

Anthracyclines (AC) are antitumor antibiotics with significant activity against solid and hematologic malignancies. One problem preventing more widespread use has been the development of cardiac toxicity. Experimental evidence supports oxidant stress as an important trigger and/or mediator of AC-induced cardiotoxicity (ACT). Therefore, reducing oxidant stress should be protective against ACT. To determine whether antioxidant protein overexpression can reduce ACT, we developed a cell culture model system using the H9C2 cardiac cell line exhibiting controlled overexpression of the α4-isoform of glutathione- S-transferase (GST). Treatment with the AC doxorubicin (DOX) produced both oncosis, manifested by an increase in the number of cells staining positive for Trypan blue, and apoptosis, indicated by the presence of positive terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. In both cases, the loss of cell viability was preceded by an AC-induced increase in fluorescence with carboxy-2′,7′-dichlorofluorescein diacetate, demonstrating the presence of high levels of reactive oxygen species (ROS). The DOX-induced increase in ROS was reduced to control levels by maximal GST overexpression. Coincident with this elimination of oxidative stress, there was a reduction in both Trypan blue and TUNEL-positive cells, indicating that GST overexpression reduced both ROS and cell death in this model system. We conclude that GST overexpression may be an important part of a protective strategy against ACT and that this model system will aid in defining steps in the pathway(s) leading to AC-induced cell death that can be therapeutically manipulated.
Characterization of embryonic cardiac pacemaker and atrioventricular conduction physiology in Xenopus laevis using noninvasive imaging
Authors: Bartlett Heather L. , Scholz Thomas D. , Lamb Fred S. , Weeks Daniel L.
Published: May 2004

Congenital heart defects often include altered conduction as well as morphological changes. Model organisms, like the frog Xenopus laevis, offer practical advantages for the study of congenital heart disease. X. laevis embryos are easily obtained free living, and the developing heart is readily visualized. Functional and morphological evidence for a conduction system is available for adult frog hearts, but information on the normal properties of embryonic heart contraction is lacking, especially in intact animals. With the use of fine glass microelectrodes, we were able to obtain cardiac recordings and make standard electrophysiological measurements in 1-wk-old embryos ( stage 46). In addition, a system using digital analysis of video images was adapted for measurement of the standard cardiac intervals and compared with invasive measurements. Video images were obtained of the heart in live, pharmacologically paralyzed, stage 46 X. laevis embryos. Normal values for the timing of the cardiac cycle were established. Intervals determined by video analysis ( n = 53), including the atrial and ventricular cycle lengths (473 ± 10 ms and 464 ± 19 ms, respectively) and the atrioventricular interval (169 ± 5 ms) were not statistically different from those determined by intrathoracic cardiac recordings. We also present the data obtained from embryos treated with standard medications that affect the human conduction system. We conclude that the physiology of embryonic X. laevis cardiac conduction can be noninvasively studied by using digital video imaging. Additionally, we show the response of X. laevis embryonic hearts to chronotropic agents is similar but not identical to the response of the human heart.
Remodeling of the chronic severely failing ischemic sheep heart after coronary microembolization: functional, energetic, structural, and cellular responses
Authors: Huang Yifei , Hunyor Stephen N. , Jiang Lele , Kawaguchi Osamu , Shirota Kazuaki , Ikeda Yoshihiko , Yuasa Takeshi , Gallagher Gabrielle , Zeng Biao , Zheng Xing
Published: May 2004

The mandatory use of pharmacotherapy in human heart failure (HF) impedes further study of natural history and remodeling mechanisms. We created a sheep model of chronic, severe, ischemic HF [left ventricular (LV) ejection fraction (LVEF) <35% stable over 4 wk] by selective coronary microembolization under general anesthesia and followed hemodynamic, energetic, neurohumoral, structural, and cellular responses over 6 mo. Thirty-eight sheep were induced into HF (58% success), with 23 sheep followed for 6 mo (21 sheep with sufficient data for analysis) after the LVEF stabilized (median of 3 embolizations). Early doubling of LV end-diastolic pressure persisted, as did increases in LV end-diastolic volume, LV wall stress, and LV wall thinning. Contractile impairment (LV end-systolic elastance, LV preload recruitable stroke work, and dobutamine-responsive contractile reserve) and diastolic dysfunction also remained stable. Cardiac mechanical energy efficiency did not recover. Plasma atrial natriuretic peptide levels remained elevated, but rises in plasma aldosterone and renin activity were transient. Collagen content increased 170%, the type I-to-III phenotype ratio doubled in the LV, but right ventricular collagen remained unaltered. Fas ligand cytokine levels correlated with expression of both caspase-3 and -2, suggesting a link in the apoptotic “death cascade.” Caspase-3 activity also bore a close relationship to LV meridional wall stress calculated from echocardiographic and intraventricular pressure measurements. We concluded that the stability of chronic untreated severe ischemic HF depends on the recruitment of myocardial remodeling mechanisms that involve an interaction among hemodynamic load, contractile efficiency/energetics, neurohumoral activation, response of the extracellular matrix, wall stress, and the myocyte apoptotic pathway.
Expressions of adrenomedullin mRNA and protein in rats with hypobaric hypoxia-induced pulmonary hypertension
Authors: Nakanishi Kuniaki , Osada Hiroshi , Uenoyama Maki , Kanazawa Fumiko , Ohrui Nobuhiro , Masaki Yoshinori , Hayashi Takuya , Kanatani Yasuhiro , Ikeda Tomosumi , Kawai Toshiaki
Published: May 2004

Experimental pulmonary hypertension induced in a hypobaric hypoxic environment (HHE) is characterized by structural remodeling of the heart and pulmonary arteries. Adrenomedullin (AM) has diuretic, natriuretic, and hypotensive effects. To study the possible effects of HHE on the AM synthesis system, 150 male Wistar rats were housed in a chamber at the equivalent of a 5,500-m altitude level for 21 days. After 14 days of exposure to HHE, pulmonary arterial pressure (PAP) was significantly increased (compared with control rats). The plasma AM protein level was significantly increased on day 21 of exposure to HHE. In the right ventricle (RV), right atrium, and left atrium of the heart, the expressions of AM mRNA and protein were increased in the middle to late phase (5–21 days) of HHE, whereas in the brain and lung they were increased much earlier (0.5–5 days). In situ hybridization and immunohistochemistry showed AM mRNA and protein staining to be more intense in the RV in animals in the middle to late phase of HHE exposure than in the controls. During HHE, these changes in AM synthesis, which occurred strongly in the RV, occurred alongside the increase in PAP. Conceivably, AM may play a role in modulating pulmonary hypertension in HHE.
Reduced release of nitric oxide to shear stress in mesenteric arteries of aged rats
Authors: Sun Dong , Huang An , Yan Ellen H. , Wu Zhiping , Yan Changdong , Kaminski Pawel M. , Oury Tim D. , Wolin Michael S. , Kaley Gabor
Published: May 2004

We hypothesized that aging is characterized by a reduced release of nitric oxide (NO) in response to shear stress in resistance vessels. Mesenteric arterioles and arteries of young (6 mo) and aged (24 mo) male Fischer 344 rats were isolated and cannulated. Shear stress (15 dyn/cm2)-induced dilation was significantly reduced and shear stress (1, 5, 10, and 15 dyn/cm2)-induced increases in perfusate nitrite were significantly smaller at all shear stress levels in vessels of aged rats. Inhibition of NO synthesis abolished shear stress-induced release of nitrite. Furthermore, shear stress (15 dyn/cm2)-induced release of nitrate was significantly higher and total nitrite (nitrite plus nitrate) was significantly lower in vessels of aged rats. Tiron or SOD significantly increased nitrite released from vessels of aged rats, but this was still significantly less than that in young rats. Superoxide production was increased and the activity of SOD was decreased in vessels of aged rats. There were no differences in endothelial NO synthase (eNOS) protein and basal activity or in Cu/Zn-SOD and Mn-SOD proteins in vessels of the two groups, but extracellular SOD was significantly reduced in vessels of aged rats. Maximal release of NO induced by shear stress plus ACh (10−5 M) was comparable in the two groups, but phospho-eNOS in response to shear stress (15 dyn/cm2) was significantly reduced in vessels of aged rats. These data suggest that an increased production of superoxide, a reduced activity of SOD, and an impaired shear stress-induced activation of eNOS are the causes of the decreased shear stress-induced release of NO in vessels of aged rats.
Myocardial carnitine palmitoyltransferase I expression and long-chain fatty acid oxidation in fetal and newborn lambs
Authors: Bartelds Beatrijs , Takens Janny , Smid Gioia B. , Zammit Victor A. , Prip-Buus Carina , Kuipers Jaap R. G. , van der Leij Feike R.
Published: May 2004

Carnitine palmitoyltransferase I (CPT I) catalyzes the conversion of acyl-CoA to acylcarnitine at the outer mitochondrial membrane and is a key enzyme in the control of long-chain fatty acid (LC-FA) oxidation. Because myocardial LC-FA oxidation increases dramatically after birth, we determined the extent to which CPT I expression contributes to these changes in the perinatal lamb. We measured the steady-state level of transcripts of the CPT1A and CPT1B genes, which encode the liver (L-CPT I) and muscle CPT I (M-CPT I) isoforms, respectively, as well as the amount of these proteins, their total activity, and the amount of carnitine in left ventricular tissue from fetal and newborn lambs. We compared these data with previously obtained myocardial FA oxidation rates in vivo in the same model. The results showed that CPT1B was already expressed before birth and that total CPT I expression transiently increased after birth. The protein level of M-CPT I was high throughout development, whereas that of L-CPT I was only transiently upregulated in the first week after birth. The total CPT I activity in vitro also increased after birth. However, the increase in myocardial FA oxidation measured in vivo (112-fold) by far exceeded the increase in gene expression (2.2-fold), protein amount (1.1-fold), and enzyme activity (1.2-fold) in vitro. In conclusion, these results stress the importance of substrate supply per se in the postnatal increase in myocardial FA oxidation. M-CPT I is expressed throughout perinatal development, making it a primary target for metabolic modulation of myocardial FA oxidation.
Atorvastatin completely inhibits VEGF-induced ACE upregulation in human endothelial cells
Authors: Saijonmaa Outi , Nyman Tuulikki , Stewen Pia , Fyhrquist Frej
Published: May 2004

Angiotensin-converting enzyme (ACE) plays an important role in the pathophysiology of cardiovascular disease. We investigated whether atorvastatin, a powerful agent for the prevention and treatment of cardiovascular disease, influences ACE production in endothelial cells. Human umbilical cord vein endothelial cells were treated with VEGF (476 pM), which induced ACE upregulation. Cotreatment with atorvastatin (0.1–10 μM) dose dependently inhibited VEGF-induced ACE upregulation. In the presence of mevalonate (100 μM), atorvastatin failed to downregulate VEGF-induced ACE production. Cotreatment of the cells with either farnesylpyrophosphate (FPP; 5 μM) or geranylgeranylpyrophosphate (GGPP; 5 μM) partially inhibited the suppressive effect of atorvastatin. Pretreatment of the cells with Rho-associated protein kinase inhibitor, Y-27632 (10 μM), partially inhibited VEGF-induced ACE upregulation. VEGF (476 pM) caused PKC phosphorylation, which was inhibited by cotreatment of the cells with atorvastatin. Atorvastatin inhibited VEGF-induced ACE upregulation probably by inhibiting PKC phosphorylation. This effect was mediated via inhibition of the mevalonate pathway. ACE downregulation may be an additional beneficial effect of statins in the treatment of cardiovascular disease.
Left atrial conduit volume is generated by deviation from the constant-volume state of the left heart: a combined MRI-echocardiographic study
Authors: Bowman Andrew W. , Kovács Sándor J.
Published: May 2004

Although modeling the four-chambered heart as a constant-volume pump successfully predicts causal physiological relationships between cardiac indexes previously deemed unrelated, the real four-chambered heart slightly deviates from the constant-volume state by ventricular end systole. This deviation has consequences that affect chamber function, specifically, left atrial (LA) function. LA attributes have been characterized as booster pump, reservoir, and conduit functions, yet characterization of their temporal occurrence or their causal relationship to global heart function has been lacking. We investigated LA function in the context of the constant-volume attribute of the left heart in 10 normal subjects using cardiac magnetic resonance imaging (MRI) and contemporaneous Doppler echocardiography synchronized via ECG. Left ventricular (LV) and LA volumes as a function of time were determined via MRI. Transmitral flow, pulmonary vein (PV) flow, and lateral mitral annular velocity were recorded via echocardiography. The relationship between the MRI-determined diastolic LA conduit-volume (LACV) filling rate and systolic LA filling rate correlate well with the relationship between the echocardiographically determined average flow rate during the early portion of the PV D wave and the average flow rate during the PV S wave ( r = 0.76). We conclude that the end-systolic deviation from constant volume for the left heart requires the generation of the LACV during diastole. Because early rapid filling of the left ventricle is the driving force for LACV generation while the left atrium remains passive, it may be more appropriate to consider LACV to be a property of ventricular diastolic rather than atrial function.
Transmural mechanics at left ventricular epicardial pacing site
Authors: Ashikaga Hiroshi , Omens Jeffrey H. , Ingels Neil B. , Covell James W.
Published: May 2004

Left ventricular (LV) epicardial pacing acutely reduces wall thickening at the pacing site. Because LV epicardial pacing also reduces transverse shear deformation, which is related to myocardial sheet shear, we hypothesized that impaired end-systolic wall thickening at the pacing site is due to reduction in myocardial sheet shear deformation, resulting in a reduced contribution of sheet shear to wall thickening. We also hypothesized that epicardial pacing would reverse the transmural mechanical activation sequence and thereby mitigate normal transmural deformation. To test these hypotheses, we investigated the effects of LV epicardial pacing on transmural fiber-sheet mechanics by determining three-dimensional finite deformation during normal atrioventricular conduction and LV epicardial pacing in the anterior wall of normal dog hearts in vivo. Our measurements indicate that impaired end-systolic wall thickening at the pacing site was not due to selective reduction of sheet shear, but rather resulted from overall depression of fiber-sheet deformation, and relative contributions of sheet strains to wall thickening were maintained. These findings suggest lack of effective end-systolic myocardial deformation at the pacing site, most likely because the pacing site initiates contraction significantly earlier than the rest of the ventricle. Epicardial pacing also induced reversal of the transmural mechanical activation sequence, which depressed sheet extension and wall thickening early in the cardiac cycle, whereas transverse shear and sheet shear deformation were not affected. These findings suggest that normal sheet extension and wall thickening immediately after activation may require normal transmural activation sequence, whereas sheet shear deformation may be determined by local anatomy.
Acute myocardial infarction induces hypothalamic cytokine synthesis
Authors: Francis Joseph , Chu Yi , Johnson Alan Kim , Weiss Robert M. , Felder Robert B.
Published: May 2004

The inflammatory milieu of acute myocardial infarction (MI) is theoretically conducive to enhanced cytokine synthesis within the brain. We tested the hypothesis that synthesis of tumor necrosis factor-α (TNF-α), an indicator of proinflammatory cytokine activity, increases in brain after MI. MI was induced in rats by ligating the left anterior descending coronary artery and confirmed by echocardiography. Plasma and tissue levels of TNF-α were measured using ELISA; TNF-α mRNA was measured with real-time PCR. Heart, brain, and plasma samples were obtained 0.5, 1, 4, or 24 h or 4 wk after MI. TNF-α synthesis increased in the brain, heart, and plasma within minutes to hours after MI and was sustained over the interval tested. Among the brain tissues examined, TNF-α increased selectively in hypothalamus. Chronic treatment with pentoxifylline prevented the increases in TNF-α in brain, heart, and plasma measured 4 wk after MI. MI-induced cytokine synthesis in the hypothalamus and its prevention by pentoxifylline have important implications in the context of the development of heart failure after MI.
Distribution of stress and strain along the porcine aorta and coronary arterial tree
Authors: Guo Xiaomei , Kassab Ghassan S.
Published: May 2004

The existence of a homeostatic state of stresses and strains has been axiomatic in the cardiovascular system. The objective of this study was to determine the distribution of circumferential stress and strain along the aorta and throughout the coronary arterial tree to test this hypothesis. Silicone elastomer was perfused through the porcine aorta and coronary arterial tree to cast the arteries at physiological pressure. The loaded and zero-stress dimensions of the vessels were measured. The aorta (1.8 cm) and its secondary branches were considered down to 1.5 mm diameter. The left anterior descending artery (4.5 mm) and its branches down to 10 μm were also measured. The Cauchy mean circumferential stress and midwall stretch ratio were calculated. Our results show that the stretch ratio and Cauchy stress were lower in the thoracic than in the abdominal aorta and its secondary branches. The opening angle (θ) and midwall stretch ratio (λ) showed a linear variation with order number ( n) as follows: θ = 10.2 n + 63.4 ( R2 = 0.989) and λ = 4.47 × 10−2 n + 1.1 ( R2 = 0.995). Finally, the stretch ratio and stress varied between 1.2 and 1.6 and between 10 and 150 kPa, respectively, along the aorta and left anterior descending arterial tree. The relative uniformity of strain (50% variation) from the proximal aorta to a 10-μm arteriole implies that the vascular system closely regulates the degree of deformation. This suggests a homeostasis of strain in the cardiovascular system, which has important implications for mechanotransduction and for vascular growth and remodeling.
Red blood cell dysfunction in septic glucose-6-phosphate dehydrogenase-deficient mice
Authors: Spolarics Zoltán , Condon Michael R. , Siddiqi Muhammad , Machiedo George W. , Deitch Edwin A.
Published: May 2004

Glucose-6-phosphate dehydrogenase (G-6-PDH) deficiency is the most common known human genetic polymorphism. This study tested the hypothesis that G-6-PDH deficiency worsens sepsis-induced erythrocyte dysfunction. Sepsis (24 h) was induced by cecal ligation and puncture in wild-type (WT) and G-6-PDH-deficient (G-6-PDH activity 15% of WT) mice. Erythrocyte responses were tested in whole blood as well as in subpopulations of circulating erythrocytes. Whereas erythrocyte deformability was similar in unchallenged deficient and WT animals, sepsis decreased erythrocyte deformability that was more pronounced in deficient than WT animals. Sepsis also resulted in anemia and hemolysis in deficient compared with WT animals. Mean corpuscular hemoglobin content and erythrocyte deformability decreased in younger erythrocyte subpopulations from septic deficient compared with WT animals. Sepsis decreased the reduced-to-oxidized glutathione ratio in erythrocytes from both deficient and WT animals; however, plasma glutathione increased more in deficient than in WT animals. Erythrocyte content of band 3 associated with the cytoskeleton was elevated in deficient compared with WT erythrocytes. The antioxidant N-acetyl-l-cysteine in vivo alleviated the sepsis-induced decrease in erythrocyte deformability in deficient animals compared with sham-operated control animals. This study demonstrates that a mild degree of G-6-PDH deficiency (comparable to the human class III G-6-PDH deficiencies) worsens erythrocyte dysfunction during sepsis. Increased erythrocyte rigidity and tendency for hemolysis together with alterations in band 3-spectrin interactions may contribute to the immunomodulatory effects of G-6-PDH deficiency observed after major trauma and infections in humans.
Limited transfer of cytosolic NADH into mitochondria at high cardiac workload
Authors: O'Donnell J. Michael , Kudej Raymond K. , LaNoue Kathyrn F. , Vatner Stephen F. , Lewandowski E. Douglas
Published: May 2004

Glycolysis supplements energy synthesis at high cardiac workloads, producing not only ATP but also cytosolic NADH and pyruvate for oxidative ATP synthesis. Despite adequate Po2, speculation exists that not all cytosolic NADH is oxidized by the mitochondria, leading to lactate production. In this study, we elucidate the mechanism for limited cytosolic NADH oxidation and increased lactate production at high workload despite adequate myocardial blood flow and oxygenation. Reducing equivalents from glycolysis enter mitochondria via exchange of mitochondrial α-ketoglutarate (α-KG) for cytosolic malate. This exchange was monitored at baseline and at high workloads by comparing13C enrichment between the products of α-KG oxidation (succinate) and α-KG efflux from mitochondria (glutamate). Under general anesthesia, a left thoracotomy was performed on 14 dogs and [2-13C]acetate was infused into the left anterior descending artery for 40 min. The rate-pressure product was 9,035 ± 1,972 and 21,659 ± 5,266 mmHg·beats·min−1( n = 7) at baseline ( n = 7) and with dobutamine, respectively.13C enrichment of succinate was 57 ± 10% at baseline and 45 ± 13% at elevated workload (not significant), confirming oxidation of [2-13C]acetate. However, cytosolic glutamate enrichment, a marker of cytosolic NADH transfer to mitochondria, was dramatically reduced at high cardiac workload (11 ± 1%) vs. baseline (50 ± 14%, P < 0.05). This reduced exchange of13C from α-KG to cytosolic glutamate at high work indicates reduced shuttling of cytosolic reducing equivalents into the mitochondria. Myocardial tissue lactate increased 78%, countering this reduced oxidation of cytosolic NADH. The findings elucidate a contributing mechanism to glycolysis outpacing glucose oxidation in the absence of myocardial ischemia.
Nitric oxide attenuates insulin- or IGF-I-stimulated aortic smooth muscle cell motility by decreasing H2O2levels: essential role of cGMP
Authors: Zhuang Daming , Ceacareanu Alice-Corina , Lin Yi , Ceacareanu Bogdan , Dixit Madhulika , Chapman Kenneth E. , Waters Christopher M. , Rao Gadiparthi N. , Hassid Aviv
Published: May 2004

Insulin and insulin-like growth factor I (IGF-I) both play important roles in vascular remodeling. Moreover, nitric oxide (NO) is well established as a counterregulatory agent that opposes the actions of several vascular agonists, in part by decreasing smooth muscle motility. We tested the hypothesis that NO blocks insulin or IGF-I-induced rat aortic smooth muscle cell motility via a mechanism involving the attenuation of agonist-induced elevation of hydrogen peroxide levels and cGMP as mediator. Insulin or IGF-I induced an increase of hydrogen peroxide levels and cell motility. Both effects were blocked by catalase or diphenyleneiodonium, indicating that hydrogen peroxide elevation is necessary for induction of cell motility. Two NO donors mimicked the effects of catalase, indicating that NO decreases cell motility by suppressing agonist-induced elevation of hydrogen peroxide. A cGMP analogue mimicked the effect of NO, whereas a guanyl cyclase inhibitor blocked the effect of NO on hydrogen peroxide levels, indicating that elevation of cGMP is both necessary and sufficient to account for the reduction of hydrogen peroxide levels. A NO donor as well as a cGMP analogue attenuated insulin-stimulated NADPH activity, indicating that NO decreases hydrogen peroxide levels by inhibiting the generation of superoxide, via a cGMP-mediated mechanism. Finally, exogenous hydrogen peroxide increased cell motility and reversed the inhibitory effect of cGMP. These results support the view that NO plays an antioxidant role via reduction of hydrogen peroxide in cultured rat aortic smooth muscle cells and that this effect is both necessary and sufficient to account for its capacity to decrease cell motility.
Cerebral artery reactivity changes during pregnancy and the postpartum period: a role in eclampsia?
Authors: Cipolla Marilyn J. , Vitullo Lisa , McKinnon John
Published: May 2004

Eclampsia is thought to be similar to hypertensive encephalopathy, whereby acute elevations in intravascular pressure cause forced dilatation (FD) of intrinsic myogenic tone of cerebral arteries and arterioles, decreased cerebrovascular resistance, and hyperperfusion. In the present study, we tested the hypothesis that pregnancy and/or the postpartum period predispose cerebral arteries to FD by diminishing pressure-induced myogenic activity. We compared the reactivity to pressure (myogenic activity) as well as factors that modulate the level of tone of third-order branches (<200 μm) of the posterior cerebral artery (PCA) that were isolated from nonpregnant (NP, n = 7), late-pregnant (LP, 19 days, n = 10), and postpartum (PP, 3 days, n = 8) Sprague-Dawley rats under pressurized conditions. PCAs from all groups of animals developed spontaneous tone within the myogenic pressure range (50–150 mmHg) and constricted arteries at 100 mmHg (NP, 30 ± 3; LP, 39 ± 4; and PP, 42 ± 7%; P > 0.05). This level of myogenic activity was maintained in the NP arteries at all pressures; however, both LP and PP arteries dilated at considerably lower pressures compared with NP, which lowered the pressure at which FD occurred from >175 for NP to 146 ± 6.5 mmHg for LP ( P < 0.01 vs. NP) and 162 ± 7.7 mmHg for PP ( P < 0.01 vs. NP). The amount of myogenic tone was also significantly diminished at 175 mmHg compared with NP: percent tone for NP, LP, and PP animals were 35 ± 2, 11 ± 3 ( P < 0.01 vs. NP), and 20 ± 7% ( P < 0.01 vs. NP), respectively. Inhibition of nitric oxide (NO) with 0.1 mM Nω-nitro-l-arginine (l-NNA) caused constriction of all vessel types that was significantly increased in the PP arteries, which demonstrates significant basal NO production. Reactivity to 5-hydroxytryptamine (serotonin) was assessed in the presence of l-NNA and indomethacin. There was a differential response to serotonin: PCAs from NP animals dilated, whereas LP and PP arteries constricted. These results suggest that both pregnancy and the postpartum period predispose the cerebral circulation to FD at lower pressures, a response that may lower cerebrovascular resistance and promote hyperperfusion when blood pressure is elevated, as occurs during eclampsia.
Role of AT1 receptors and NAD(P)H oxidase in diabetes-aggravated ischemic brain injury
Authors: Kusaka Ikuyo , Kusaka Gen , Zhou Changman , Ishikawa Mami , Nanda Anil , Granger D. Neil , Zhang John H. , Tang Jiping
Published: May 2004

The objective of the present study was to examine the role of the angiotensin II type 1 receptor (AT1-R) in the diabetes-aggravated oxidative stress and brain injury observed in a rat model of combined diabetes and focal cerebral ischemia. Diabetes was induced by an injection of streptozotoxin (STZ; 55 mg/kg iv) at 8 wk of age. Two weeks after the induction of diabetes, some animals received continuous subcutaneous infusion of the AT1-R antagonist candesartan (0.5 mg·kg−1·day−1) for 14 days. Focal cerebral ischemia, induced by middle cerebral artery occlusion/reperfusion (MCAO), was conducted at 4 wk after STZ injection. Male Sprague-Dawley rats ( n = 189) were divided into five groups: normal control, diabetes, MCAO, diabetes + MCAO, and diabetes + MCAO + candesartan. The major observations were that 1) MCAO produced typical cerebral infarction and neurological deficits at 24 h that were accompanied by elevation of NAD(P)H oxidase gp91phox and p22phox mRNAs, and lipid hydroperoxide production in the ipsilateral hemisphere; 2) diabetes enhanced NAD(P)H oxidase gp91phox and p22phox mRNA expression, potentiated lipid peroxidation, aggravated neurological deficits, and enlarged cerebral infarction; and 3) candesartan reduced the expression of gp91phox and p22phox, decreased lipid peroxidation, lessened cerebral infarction, and improved the neurological outcome. We conclude that diabetes exaggerates the oxidative stress, NAD(P)H oxidase induction, and brain injury induced by focal cerebral ischemia. The diabetes-aggravated brain injury involves AT1-Rs. We have shown for the first time that candesartan reduces brain injury in a combined model of diabetes and cerebral ischemia.
Transmembrane action potential heterogeneity in the canine isolated arterially perfused right atrium: effect ofIKrandIKur/Itoblock
Authors: Burashnikov Alexander , Mannava Sandeep , Antzelevitch Charles
Published: May 2004

The role of electrical heterogeneity in development of cardiac arrhythmias is well recognized. The extent to which transmembrane action potential (TAP) heterogeneity contributes to the normal electrophysiology of well-oxygenated atria is not well defined. The principal objective of the present study was to define regional and transmural differences in characteristics of the TAP in isolated superfused and arterially perfused canine right atrial (RA) preparations under baseline, rapidly activating delayed rectifier K+current ( IKr) block, and combined block of ultrarapid delayed rectifier and transient outward K+current ( IKur/ Itoblock). Superfused preparations that survived generally displayed a triangle-shaped TAP. Exceptions included cells from the crista terminalis, where TAPs with a normal plateau could be recorded. In contrast, most TAPs recorded from throughout the perfused RA displayed a spike-and-dome and/or plateau morphology. The perfused RA displayed a heterogeneous distribution of repolarization, Vmax, and spike-and-dome morphology along the epicardial and endocardial surfaces as well as transmurally, in the region of the upper crista terminalis. IKrblock with E-4031 prolonged repolarization homogeneously in the perfused RA, whereas IKur/ Itoblock using low concentrations of 4-aminopyridine abbreviated action potential duration at 90% repolarization heterogeneously, leading to a reduction in dispersion of repolarization. Our data indicate that the electrical heterogeneities, previously described for the canine ventricle, also exist within the atria and that IKrblock does not accentuate and IKur/ Itoblock reduces RA dispersion of repolarization. Our study also points to major differences in the transmembrane activity recorded using superfused vs. arterially perfused atrial preparations.
Patency of the preterm fetal ductus arteriosus is regulated by endothelial nitric oxide synthase and is independent of vasa vasorum in the mouse
Authors: Richard Charissa , Gao Ju , LaFleur Bonnie , Christman Brian W. , Anderson Judy , Brown Naoko , Reese Jeff
Published: May 2004

Patency of the fetal ductus arteriosus (DA) is maintained in an environment of low relative oxygen tension and a preponderance of vasodilating forces. In addition to prostaglandins, nitric oxide (NO), a potent vasodilator in the pulmonary and systemic vasculatures, has been implicated in regulation of the fetal DA. To further define the contribution of NO to DA patency, the expression and function of NO synthase (NOS) isoforms were examined in the mouse DA on days 17–19 of pregnancy and after birth. Our results show that endothelial NOS (eNOS) is the predominant isoform expressed in the mouse DA and is localized in the DA endothelium by in situ hybridization. Despite rapid constriction of the DA after birth, eNOS expression levels were unchanged throughout the fetal and postnatal period. Pharmacological inhibition of prostaglandin vs. NO synthesis in vivo showed that the preterm fetal DA on day 16 is more sensitive to NOS inhibition than the mature fetal DA on day 19, whereas prostaglandin inhibition results in marked DA constriction on day 19 but minimal effects on the day 16 DA. Combined prostaglandin and NO inhibition caused additional DA constriction on day 16. The contribution of vasa vasorum to DA regulation was also examined. Immunoreactive platelet endothelial cell adhesion molecule and lacZ tagged FLK1 localized to DA endothelial cells but revealed the absence of vasa vasorum within the DA wall. Similarly, there was no evidence of vasa vasorum by vascular casting. These studies indicate that eNOS is the primary source of NO in the mouse DA and that vasomotor tone of the preterm fetal mouse DA is regulated by eNOS-derived NO and is potentiated by prostaglandins. In contrast to other species, mechanisms for DA patency and closure appear to be independent of any contribution of the vasa vasorum.
Estrogen receptor-α expression in osmosensitive elements of the lamina terminalis: regulation by hypertonicity
Authors: Somponpun Suwit J. , Johnson Alan Kim , Beltz Terry , Sladek Celia D.
Published: May 2004

The subfornical organ (SFO), median preoptic nucleus (MnPO), and organum vasculosum lamina terminalis (OVLT), which are associated with the lamina terminalis, are important in the control of body fluid balance. Neurons in these regions express estrogen receptor (ER)-α, but whether the ER-α neurons are activated by hypertonicity and whether hypertonicity regulates ER-α expression are not known. Using fluorescent, double-label immunocytochemistry, we examined the expression of ER-α-immunoreactivity (ir) and Fos-ir in control and water-deprived male rats. In control animals, numerous ER-α-positive neurons were expressed in the periphery of the SFO, in both the dorsal and ventral MnPO, and in the dorsal cap of the OVLT. Fos-positive neurons were sparse in euhydrated rats but were numerous in the SFO, MnPO, and the dorsal cap of the OVLT after 48-h water deprivation. Most ER-α-ir neurons in these areas were positive for Fos, indicating a significant degree of colocalization. To examine the effect of dehydration on ER-α expression, animals with and without lesions surrounding the anterior and ventral portion of the 3rd ventricle (AV3V) were water deprived for 48 h. Water deprivation resulted in a moderate increase in ER-α-ir in the SFO of sham-lesioned rats ( P = 0.03) and a dramatic elevation in AV3V-lesioned animals ( P < 0.05). This was probably induced by the significant increase in plasma osmolality in both dehydrated groups ( P < 0.001) rather than a decrease in blood volume, because hematocrit was significantly increased only in the dehydrated sham-lesioned animals. Thus these studies implicate the osmosensitive regions of the lamina terminalis as possible targets for sex steroid effects on body fluid homeostasis.
Prenatal exposure to ethanol causes partial diabetes insipidus in adult rats
Authors: Knee Daniel S. , Sato Aileen K. , Uyehara Catherine F. T. , Claybaugh John R.
Published: May 2004

Chronic consumption of ethanol in adult rats and humans leads to reduced AVP-producing neurons, and prenatal ethanol (PE) exposure has been reported to cause changes in the morphology of AVP-producing cells in the suprachiasmatic nucleus of young rats. The present studies further characterize the effects of PE exposure on AVP in the young adult rat, its hypothalamic synthesis, pituitary storage, and osmotically stimulated release. Pregnant rats were fed a liquid diet with 35% of the calories from ethanol or a control liquid diet for days 7–22 of pregnancy. Water consumption and urine excretion rate were measured in the offspring at 60–68 days of age. Subsequently, the offspring were infused with 5% NaCl at 0.05 ml·kg−1·min−1 with plasma samples taken before and at three 40-min intervals during infusion for measurement of AVP and osmolality. Urine output and water intake were ∼20% greater in PE-exposed rats than in rats with no PE exposure, and female rats had a greater water intake than males. The relationship between plasma osmolality and AVP in PE-exposed rats was parallel to, but shifted to the right of, the control rats, indicating an increase in osmotic threshold for AVP release. Pituitary AVP was reduced by 13% and hypothalamic AVP mRNA content was reduced by 35% in PE-exposed rats. Our data suggest that PE exposure can cause a permanent condition of a mild partial central diabetes insipidus.
Circulating free nitrotyrosine in obstructive sleep apnea
Authors: Svatikova Anna , Wolk Robert , Wang Hui H. , Otto Maria E. , Bybee Kevin A. , Singh Ravinder J. , Somers Virend K.
Published: May 2004

Obstructive sleep apnea (OSA) has been increasingly linked to cardiovascular disease, endothelial dysfunction, and oxidative stress, generated by repetitive nocturnal hypoxemia and reperfusion. Circulating free nitrotyrosine has been reported as a novel biomarker of nitric oxide (NO)-induced oxidative/nitrosative stress. Nitrosative stress has been implicated as a possible mechanism for development of cardiovascular diseases. We tested the hypothesis that repetitive severe hypoxemia resulting from OSA would increase NO-mediated oxidative stress. We studied 10 men with newly diagnosed moderate to severe OSA who were free of other diseases, had never been treated for OSA, and were taking no medications. Nitrotyrosine measurements, performed by liquid chromatography-tandem mass spectrometry, were made before and after untreated apneic sleep. We compared free nitrotyrosine levels in these patients with those obtained at similar times in 10 healthy male control subjects without OSA, with similar age and body mass index. Evening baseline nitrotyrosine levels were similar before sleep in the control and OSA groups [0.16 ± 0.01 and 0.15 ± 0.01 ng/ml, respectively, P = not significant (NS)]. Neither normal nor disturbed apneic sleep led to significant changes of plasma nitrotyrosine (morning levels: control group 0.14 ± 0.01 ng/ml; OSA group 0.15 ± 0.01 ng/ml, P = NS). OSA was not accompanied by increased circulating free nitrotyrosine either at baseline or after sleep. This observation suggests that repetitive hypoxemia during OSA does not result in increased NO-mediated oxidative/nitrosative stress in otherwise healthy subjects with OSA.
The urinary proteome in Fanconi syndrome implies specificity in the reabsorption of proteins by renal proximal tubule cells
Authors: Cutillas Pedro R. , Chalkley Robert J. , Hansen Kirk C. , Cramer Rainer , Norden Anthony G. W. , Waterfield Mike D. , Burlingame Alma L. , Unwin Robert J.
Published: May 2004

Polypeptides present in the glomerular filtrate are almost completely reabsorbed in the first segment of the proximal tubule by receptor-mediated endocytosis; in renal Fanconi syndrome (FS), there is failure to reabsorb many of these polypeptides. We have compared the urinary proteomes in patients with Dent’s disease (due to a CLC5 mutation), a form of FS, with normal subjects using three different proteomic methods. No differences in the levels of several plasma proteins were detected when standardized to total protein amounts. In contrast, several vitamin and prosthetic group carrier proteins were found in higher amounts in Dent’s urine (with respect to total protein). Similarly, complement components, apolipoproteins, and some cytokines represented a larger proportion of the Dent’s urinary proteome, suggesting that such proteins are reabsorbed more efficiently than other classes of proteins. Conversely, proteins of renal origin were found in proportionately higher amounts in normal urine. Thus the uptake of filtered vitamins, which are normally bound to their respective carrier proteins to prevent urinary losses, seems a key function of the proximal tubule; in addition, this nephron segment may also play a critical role in reabsorbing potentially cytotoxic polypeptides of plasma origin, preventing them from acting at more distal nephron sites.
CD40-induced transcriptional activation of vascular endothelial growth factor involves a 68-bp region of the promoter containing a CpG island
Authors: Lapchak Peter H. , Melter Michael , Pal Soumitro , Flaxenburg Jesse A. , Geehan Christopher , Frank Markus H. , Mukhopadhyay Debabrata , Briscoe David M.
Published: May 2004

Vascular endothelial growth factor (VEGF) is produced by several cell types in the kidney, and its expression is tightly regulated for the maintenance of normal renal physiology. Increases or decreases in its expression are associated with proteinuria and renal disease. Recently, we found that the expression of VEGF is markedly induced following interactions between CD40 ligand (CD40L) and CD40. Here, endothelial cells (EC) or Jurkat T cell lines were transiently transfected with luciferase reporter constructs under the control of the human VEGF promoter and were treated with human soluble CD40L (sCD40L). We identified a CD40-responsive 68-bp region (bp −50 to +18) of the promoter and 43 bp within this region (bp −25 to +18) that have 97% homology to a sequence of CpG dinucleotides. A computerized search revealed that the CpG region has putative binding domains for the transcriptional repressor protein methyl CpG binding protein-2 (MeCP2). In EMSA, we found that the 43-bp methylated sequence formed four complex(es) with nuclear extracts from untreated EC and reduced binding of at least one complex when nuclear lysates from sCD40L-activated EC (30 min) were used. Supershift analysis using anti-MeCP2 demonstrated that most of the complex(es) in both untreated and sCD40L-activated EC involved interactions between the 43-bp DNA and MeCP2. In addition, we found that other CpG binding proteins may also interact with this region of the promoter. Taken together, this is the first demonstration that CpG binding transcriptional repressor proteins including MeCP2 may be of importance in VEGF biology.
AVP-induced VIT32 gene expression in collecting duct cells occurs via trans-activation of a CRE in the 5′-flanking region of the VIT32 gene
Authors: Thomas Christie P. , Loftus Randy W. , Liu Kang Z.
Published: May 2004

VIT32, a vasopressin-induced transcript, inhibits Na+ transport when coexpressed with the epithelial sodium channel in Xenopus laevis oocytes ( EMBO J 21: 5109–5117, 2002). To understand the mechanism of VIT32 gene regulation, we examined the effect of DDAVP and cAMP stimulation on VIT32 expression in M-1 mouse collecting duct cells and in H441 human airway epithelial cells. Elevation of cAMP with forskolin and IBMX increased VIT32 gene expression with a peak effect at 2 h. The increase in gene expression was abolished by H89 and by actinomycin D, suggesting that cAMP stimulates VIT32 mRNA expression by a PKA-mediated increase in gene transcription. An ∼1.5-kb fragment of the 5′-flanking region of VIT32 was cloned and was able to confer cAMP-stimulated reporter gene activity when transfected into M-1 and H441 cells. By deletion analysis and site-directed mutagenesis, a cAMP response element (CRE) was identified within the proximal promoter region that was sufficient to account for the increase in VIT32 gene expression seen with DDAVP and elevation of cAMP. Furthermore, DDAVP-stimulated VIT32 promoter-reporter activity was inhibited by H89 and by a dominant negative CREB construct. Finally, we were able to identify CREB as a nuclear protein that bound to the VIT32 CRE in gel mobility shift assays. In summary, DDAVP stimulates transcription of VIT32 via a CRE within the proximal promoter region of the VIT32 gene.
A naturally occurring human Nedd4–2 variant displays impaired ENaC regulation in Xenopus laevis oocytes
Authors: Fouladkou Fatemeh , Alikhani-Koopaei Rasoul , Vogt Bruno , Flores Sandra Y. , Malbert-Colas Laurence , Lecomte Marie-Christine , Loffing Johannes , Frey Felix J. , Frey Brigitte M. , Staub Olivier
Published: May 2004

The epithelial Na+ channel (ENaC) is regulated by the ubiquitin-protein ligase Nedd4–2 via interaction with ENaC PY-motifs. These PY-motifs are mutated/deleted in Liddle's syndrome, resulting in elevated Na+ reabsorption and hypertension explained partly by impaired ENaC-Nedd4–2 interaction. We hypothesized that Nedd4–2 is a susceptibility gene for hypertension and screened 856 renal patients and healthy controls for mutations in a subset of exons of the human Nedd4–2 gene that are relevant for ENaC regulation by PCR/single-strand conformational polymorphism. Several variants were identified, and one nonsynonymous mutation (Nedd4–2-P355L) was further characterized. This mutation next to the 3′ donor site of exon 15 does not affect in vitro splicing of Nedd4–2 mRNA. However, in the Xenopus oocyte expression system, Nedd4–2-P355L-dependent ENaC inhibition was weaker compared with the wild type (Nedd4–2-WT), and this difference depended on the presence of intact PY-motifs on ENaC. This could not be explained by the amount of wild type or mutant Nedd4–2 coimmunoprecipitating with ENaC. When the phosphorylation level of human Nedd4–2 Ser448 (known to be phosphorylated by the Sgk1 kinase) was determined with a specific anti-pSer448 antibody, we observed stronger basal phosphorylation of Nedd4–2-P355L. Both the phosphorylation level and the accompanying amiloride-sensitive Na+ currents could be further enhanced to approximately the same levels by coexpressing Sgk1. In addition, the role of the two other putative Sgk1 phosphorylation sites (S342 and T367) appears also to be affected by the P355L mutation. The differential phosphorylation status between wild-type and mutant Nedd4–2 provides an explanation for the different potential to inhibit ENaC activity.