bluuebunny/crossref_metadata_2025 huggingface dataset:
Caerulein-induced acute pancreatitis inhibits protein synthesis through effects on eIF2B and eIF4F
Authors: Sans Maria Dolors , DiMagno Matthew J. , D'Alecy Louis G. , Williams John A.
Published: March 2015

Acute pancreatitis (AP) has been shown in some studies to inhibit total protein synthesis in the pancreas, whereas in other studies, protein synthesis was not affected. Previous in vitro work has shown that high concentrations of cholecystokinin both inhibit protein synthesis and inhibit the activity of the guanine nucleotide exchange factor eukaryotic initiation factor (eIF)2B by increasing the phosphorylation of eIF2α. We therefore evaluated in C57BL/6 mice the effects of caerulein-induced AP on pancreatic protein synthesis, eIF2B activity and other protein translation regulatory mechanisms. Repetitive hourly injections of caerulein were administered at 50 μg/kg ip. Pancreatic protein synthesis was reduced 10 min after the initial caerulein administration and was further inhibited after three and five hourly injections. Caerulein inhibited the two major regulatory points of translation initiation: the activity of the guanine nucleotide exchange factor eIF2B (with an increase of eIF2α phosphorylation) and the formation of the eIF4F complex due, in part, to degradation of eIF4G. This inhibition was not accounted for by changes in the upstream stimulatory pathway, because caerulein activated Akt as well as phosphorylating the downstream effectors of mTOR, 4E-BP1, and ribosomal protein S6. Caerulein also decreased the phosphorylation of the eukaryotic elongation factor 2, implying that this translation factor was not inhibited in AP. Thus the inhibition of pancreatic protein synthesis in this model of AP most likely results from the inhibition of translation initiation as a result of increased eIF2α phosphorylation, reduction of eIF2B activity, and the inhibition of eIF4F complex formation.
Diurnal rhythm of H+-peptide cotransporter in rat small intestine
Authors: Pan Xiaoyue , Terada Tomohiro , Irie Megumi , Saito Hideyuki , Inui Ken-Ichi
Published: March 2015

In mammals, most physiological, biochemical, and behavioral processes show a circadian rhythm. In the present study, we examined the diurnal rhythm of the H+-peptide cotransporter (PEPT1), which transports small peptides and peptide-like drugs in the small intestine and kidney, using rats maintained in a 12-h photoperiod with free access to chow. The transport of [14C]glycylsarcosine (Gly-Sar), a typical substrate for PEPT1 by in situ intestinal loop and everted intestine, was greater in the dark phase than the light phase. PEPT1 protein and mRNA levels varied significantly, with a maximum at 2000 and minimum at 800. Similar functional and expressional diurnal variations were observed in the intestinal Na+-glucose cotransporter (SGLT1). In contrast, renal PEPT1 and SGLT1 showed little diurnal rhythmicity in protein and mRNA expression. These findings indicate that the intestinal PEPT1 undergoes diurnal regulation in its activity and expression, and this could affect the intestinal absorption of dietary protein.
Stretch-induced calcium sensitization of rat lymphatic smooth muscle
Authors: Shirasawa Yuichi , Benoit Joseph N.
Published: March 2015

The relationships between smooth muscle calcium and isometric tension generation to spontaneous lymphatic pump activity and its modulation by stretch equivalent from 0 to ∼6 cmH2O were investigated. Excised preparations of the rat thoracic duct were mounted on a wire myograph and loaded with the calcium-sensitive fluorochrome indo-1. Calcium-dependent fluorescence and isometric force were simultaneously recorded. The thoracic duct segments developed spontaneous rhythmic contractile activity. Each contraction was preceded by an increase in intracellular calcium. When the vessels were normalized and stabilized at a preload equal to 3 cmH2O, the peak generation in tension occurred 0.70 ± 0.11 s after that of calcium. Incremental stretch enhanced the frequency of the phasic activity and amplitude of isometric force generation but not the basal calcium level or the amplitude of the calcium transient. These findings suggest that stretch enhances lymphatic pump activity by increasing the pacemaker activity and the calcium sensitivity of the contractile apparatus.
5-Lipoxygenase and human pulmonary artery endothelial cell proliferation
Authors: Walker Jennifer L. , Loscalzo Joseph , Zhang Ying-Yi
Published: March 2015

Increased 5-lipoxygenase (5LO) expression in pulmonary artery endothelial cells (PAECs) has been observed in primary pulmonary hypertension, a disorder associated with pulmonary vascular remodeling and aberrant endothelial cell proliferation. To examine whether 5LO plays a role in endothelial cell proliferation, we analyzed the effect of 5LO inhibitors on cultured human PAECs. Analysis of [3H]thymidine incorporation showed that 5LO and 5LO-activating protein inhibitors AA-861, nordihydroguaiaretic acid (NDGA), and MK-886 all inhibited PAEC growth in a dose-dependent manner, with maximal inhibition of >90% and IC50values of 3.9, 1.8, and 0.48 μM, respectively. The effect of AA-861 and NDGA correlated with their effect on 5LO activity in PAECs. Concentrations of these inhibitors at or below their IC90values did not cause significant cell death as determined by lactate dehydrogenase release, but decreased cell doubling, as measured by cell counting at 24 h after serum replenishment. Analysis of DNA content suggested that the inhibitors led to an accumulation of PAECs at the G0/G1phase. Antisense oligonucleotides to 5LO mRNA delivered at a transfection efficiency of ∼60% inhibited cell growth by 40 ± 26% compared with that of a sequence-unrelated oligonucleotide. Indomethacin had no effect on PAEC growth over a range of concentrations (0.3–5 μM). These data show that 5LO inhibitors impaired the proliferative response of the cultured PAECs, suggesting that this enzyme may contribute to PAEC growth under certain pathological conditions.
Hydrogen peroxide acts as an EDHF in the piglet pial vasculature in response to bradykinin
Authors: Lacza Zsombor , Puskar Michelle , Kis Béla , Perciaccante James V. , Miller Allison W. , Busija David W.
Published: March 2015

We investigated the mechanism of EDHF-mediated dilation to bradykinin (BK) in piglet pial arteries. Topically applied BK (3 μmol/l) induced vasodilation (62 ± 12%) after the administration of N ω-nitro-l-arginine methyl ester (l-NAME) and indomethacin, which was inhibited by endothelial impairment or by the BK2 receptor antagonist HOE-140 (0.3 μmol/l). Western blotting showed the presence of BK2 receptors in brain cortex and pial vascular tissue samples. The cytochrome P-450 antagonist miconazole (20 μmol/l) and the lipoxygenase inhibitors baicalein (10 μmol/l) and cinnamyl-3,4-dyhydroxy-α-cyanocinnamate (1 μmol/l) failed to reduce the BK-induced dilation. However, the H2O2 scavenger catalase (400 U/ml) abolished the response (from 54 ± 11 to 0 ± 2 μm; P< 0.01). The ATP-dependent K+ (KATP) channel inhibitor glibenclamide (10 μmol/l) had a similar effect as well (from 54 ± 11 to 16 ± 5 μm; P < 0.05). Coapplication of the Ca2+-dependent K+ channel inhibitors charybdotoxin (0.1 μmol/l) and apamin (0.5 μmol/l) failed to reduce the response. We conclude that H2O2 mediates the non-nitric oxide-, non-prostanoid-dependent vasorelaxation to BK in the piglet pial vasculature. The response is mediated via BK2 receptors and the opening of KATP channels.
Role of glycosylation in cell surface expression and stability of HERG potassium channels
Authors: Gong Qiuming , Anderson Corey L. , January Craig T. , Zhou Zhengfeng
Published: March 2015

The human ether-à-go-go-related gene (HERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel in the heart. We previously showed that HERG channel protein is modified by N-linked glycosylation. HERG protein sequence contains two extracellular consensus sites for N-linked glycosylation (N598, N629). In this study, we used the approaches of site-directed mutagenesis and biochemical modification to inhibit N-linked glycosylation and studied the role of glycosylation in the cell surface expression and turnover of HERG channels. Our results show that N598 is the only site for N-linked glycosylation and that glycosylation is not required for the cell surface expression of functional HERG channels. In contrast, N629 is not used for glycosylation, but mutation of this site (N629Q) causes a protein trafficking defect, which results in its intracellular retention. Pulse-chase experiments show that the turnover rate of nonglycosylated HERG channel is faster than that of the glycosylated form, suggesting that N-linked glycosylation plays an important role in HERG channel stability.
Cariporide preserves mitochondrial proton gradient and delays ATP depletion in cardiomyocytes during ischemic conditions
Authors: Ruiz-Meana Marisol , Garcia-Dorado David , Pina Pilar , Inserte Javier , Agulló Luis , Soler-Soler Jordi
Published: March 2015

The mechanism by which inhibition of Na+/H+ exchanger (NHE) reduces cell death in ischemic-reperfused myocardium remains controversial. This study investigated whether cariporide could inhibit mitochondrial NHE during ischemia, delaying H+ gradient dissipation and ATP exhaustion. Mouse cardiac myocytes (HL-1) were submitted to 1 h of simulated ischemia (SI) with NaCN/deoxyglucose (pH 6.4), with or without 7 μM cariporide, and mitochondrial concentration of Ca2+ (Rhod-2), 2′, 7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and the charge difference across the mitochondrial membrane potential (Δψm, JC-1) were assessed. ATP content was measured by bioluminescence and mitochondrial swelling by spectrophotometry in isolated mitochondria. Cariporide significantly attenuated the acidification of the mitochondrial matrix induced by SI without modifying Δψm decay, and this effect was associated to a delayed ATP exhaustion and increased mitochondrial Ca2+ load. These effects were reproduced in sarcolemma-permeabilized cells exposed to SI. In these cells, cariporide markedly attenuated the fall in mitochondrial pH induced by removal of Na+ from the medium. In isolated mitochondria, cariporide significantly reduced the rate and magnitude of passive matrix swelling induced by Na+ acetate. In isolated rat hearts submitted to 40-min ischemia at different temperatures (35.5°, 37°, or 38.5°C) pretreatment with cariporide limited ATP depletion during the first 10 min of ischemia and cell death (lactate dehydrogenase release) during reperfusion. These effects were mimicked when a similar ATP preservation was achieved by hypothermia and were abolished when the sparing effect of cariporide on ATP was suppressed by hyperthermia. We conclude that cariporide acts at the mitochondrial level, delaying mitochondrial matrix acidification and delaying ATP exhaustion during ischemia. These effects can contribute to reduce cell death secondary to ischemia-reperfusion.
Effect of nitric oxide on excitatory amino acid-evoked discharge of neurons in NTS
Authors: Dias Ana Carolina Rodrigues , Colombari Eduardo , Mifflin Steven W.
Published: March 2015

N-methyl-d-aspartate (NMDA) and non-NMDA excitatory amino acid (EAA) receptor subtypes are involved in the integration of visceral afferent inputs within the nucleus of the solitary tract (NTS). Microinjection studies indicate interactions between nitric oxide (NO) and EAA receptors within the NTS. To examine these interactions at the single cell level, this study characterized the effects of the NO synthase inhibitor N G-nitro-l-arginine methyl ester (l-NAME) and the NO donor 3-[2-hydroxy-2-nitroso-1-propylhydrazino]-1-propanamine (PAPA-NONOate) on the excitatory responses of vagus nerve (VN)-evoked NTS neurons to the activation of ( RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and NMDA receptors in rats. Iontophoresis ofl-NAME did not alter spontaneous or VN-evoked discharges, but significantly decreased the number of action potentials (APs) evoked by iontophoretic application of AMPA. The effects ofl-NAME on NMDA-evoked discharge were variable; for the population, l-NAME did not change the number of APs evoked by NMDA. PAPA-NONOate enhanced the spontaneous discharge and the number of APs elicited by AMPA but not NMDA. Iontophoresis of the inactive enantiomers N G-nitro-d-arginine methyl ester and hydroxydiazenesulfonic acid 1-oxide disodium salt had no effect on AMPA-evoked discharge. Our data suggest that NO facilitates AMPA-mediated neuronal transmission within the NTS.
Conduction of hyperpolarization along hamster feed arteries: augmentation by acetylcholine
Authors: Emerson Geoffrey G. , Neild Timothy O. , Segal Steven S.
Published: March 2015

The conduction of vasodilation along resistance vessels has been presumed to reflect the electrotonic spread of hyperpolarization from cell to cell along the vessel wall through gap junction channels. However, the vasomotor response to acetylcholine (ACh) encompasses greater distances than can be explained by passive decay. To investigate the underlying mechanism for this behavior, we tested the hypothesis that ACh augments the conduction of hyperpolarization. Feed arteries ( n = 23; diameter, 58 ± 4 μm; segment length, 2–8 mm) were isolated from the hamster retractor muscle, cannulated at each end, and pressurized to 75 mmHg (at 37°C). Vessels were impaled with one or two dye-containing microelectrodes simultaneously (separation distance, 50 μm to 3.5 mm). Membrane potential ( E m) (rest, approximately −30 mV) and electrical responses were similar between endothelium and smooth muscle, as predicted for robust myoendothelial coupling. Current injection (−0.8 nA, 1.5 s) evoked hyperpolarization (−10 ± 1 mV; membrane time constant, 240 ms) that conducted along the vessel with a length constant (λ) = 1.2 ± 0.1 mm; spontaneous E m oscillations (∼1 Hz) decayed with λ = 1.2 + 0.1 mm. In contrast, ACh microiontophoresis (500 nA, 500 ms, 1 μm tip) evoked hyperpolarization (−14 ± 2 mV) that conducted with λ = 1.9 ± 0.1 mm, 60% further ( P < 0.05) than responses evoked by purely electrical stimuli. These findings indicate that ACh augments the conduction of hyperpolarization along the vessel wall.
Reactive oxygen species generated during myocardial ischemia enable energetic recovery during reperfusion
Authors: Klawitter Paul F. , Murray Holt N. , Clanton Thomas L. , Angelos Mark G.
Published: March 2015

We studied the differences between the functional and bioenergetic effects of antioxidants (AOX) administered before or after myocardial ischemia. Sprague-Dawley rat hearts were perfused with a modified Krebs-Henseleit solution and bubbled with 95% O2-5% CO2. The protocol consisted of 10 min of baseline perfusion, 20 min of global ischemia, and 30 min of reperfusion. An AOX, either 1,2-dihydroxybenzene-3,5-disulfonate (Tiron), a superoxide scavenger, or N-acetyl-l-cysteine, was infused during either baseline or reperfusion. An additional group received deferoxamine as a bolus before ischemia. Hearts were freeze-clamped at baseline, at end of ischemia, and at end of reperfusion for analysis of high-energy phosphates. All AOX, when given before ischemia, inhibited recovery of ATP compared with controls. Both Tiron and deferoxamine also inhibited recovery of phosphocreatine. AOX given before ischemia decreased the efficiency of contraction during reperfusion compared with controls. All of the changes in energetics and efficiency brought on by preischemic AOX treatment could be blocked by a preconditioning stimulus. This suggests that reactive oxygen species, which are generated during ischemia, enhance bioenergetic recovery by increasing the efficiency of contraction.
Dexrazoxane does not protect against doxorubicin-induced damage in young rats
Authors: Héon Stéphanie , Bernier Martin , Servant Nicolas , Dostanic Stevan , Wang Chunlei , Kirby Gordon M. , Alpert Lesley , Chalifour Lorraine E.
Published: March 2015

Doxorubicin (DOX), an anticancer drug, causes a dose-dependent cardiotoxicity. Some evidence suggests that female children have an increased risk for DOX-mediated cardiac damage. To determine whether the iron chelator dexrazoxane (DXR) could reduce DOX-induced cardiotoxicity in the young, we injected day 10 neonate female and male rat pups with a single dose of saline or DOX, DXR, or DXR + DOX (20:1). We followed body weight gain with growth, measured cardiac hypertrophy after a 2-wk swim exercise program, markers of apoptosis (Bcl-2, BAX, BNIP1, caspase 3 activation), oxidative stress (heme oxygenase 1, protein carbonyl levels), the chaperone protein clusterin, and the transcriptional activator early growth response gene-1 (Egr-1) in hearts of nonexercised and exercised rats on neonate day 38. All DOX-alone and DXR + DOX-treated rats showed decreased weight gain, with female rats affected earlier than male rats. DXR-alone, DOX-alone, and DXR + DOX-treated rats had an increased heart weight-to-body weight (heart wt/body wt) ratio after the exercise program with female rats showing the largest increase in heart wt/body wt. Drug-treated females also showed increased cardiac apoptosis, as measured by the increased expression of the proapoptotic proteins BAX and BNIP1 and the appearance of caspase 3 activation products, and oxidative stress, as measured by increased heme oxygenase 1 expression, and reduced Egr-1 and clusterin expression when compared with the similarly treated male rats. We conclude that DXR preinjection did not reduce DOX-induced noncardiac and cardiac damage and that young female rats were more susceptible to DXR and DOX toxicities than age-matched male rats.
MitoKATP opener, diazoxide, reduces neuronal damage after middle cerebral artery occlusion in the rat
Authors: Shimizu Katsuyoshi , Lacza Zsombor , Rajapakse Nishadi , Horiguchi Takashi , Snipes James , Busija David W.
Published: March 2015

We investigated effects of diazoxide, a selective opener of mitochondrial ATP-sensitive K+ (mitoKATP) channels, against brain damage after middle cerebral artery occlusion (MCAO) in male Wistar rats. Diazoxide (0.4 or 2 mM in 30 μl saline) or saline (sham) was infused into the right lateral ventricle 15 min before MCAO. Neurological score was improved 24 h later in the animals treated with 2 mM diazoxide (13.8 ± 0.7, n = 13) compared with sham treatment (9.5 ± 0.2, n = 6, P < 0.01). The total percent infarct volume (MCAO vs. contralateral side) of sham treatment animals was 43.6 ± 3.6% ( n = 12). Treatment with 2 mM diazoxide reduced the infarct volume to 20.9 ± 4.8% ( n = 13, P < 0.05). Effects of diazoxide were prominent in the cerebral cortex. The protective effect of diazoxide was completely prevented by the pretreatment with 5-hydroxydecanoate (100 mM in 10 μl saline), a selective blocker of mitoKATP channels ( n = 6). These results indicate that selective opening of the mitoKATP channel has neuroprotective effects against ischemia-reperfusion injury in the rat brain.
Molecular and pharmacological approaches to inhibiting nitric oxide after burn trauma
Authors: White Jean , Carlson Deborah L. , Thompson Marita , Maass David L. , Sanders Billy , Giroir Brett , Horton Jureta W.
Published: March 2015

Whereas controversial, several studies have suggested that nitric oxide (NO) alters cardiac contractility via cGMP, peroxynitrite, or poly(ADP ribose) synthetase (PARS) activation. This study determined whether burn-related upregulation of myocardial inducible NO synthase (iNOS) and NO generation contributes to burn-mediated cardiac contractile dysfunction. Mice homozygous null for the iNOS gene (iNOS knockouts) were obtained from Jackson Laboratory. iNOS knockouts (KO) as well as wild-type mice were given a cutaneous burn over 40% of the total body surface area by the application of brass probes (1 × 2 × 0.3 cm) heated to 100°C to the animals' sides and back for 5 s (iNOS/KO burn and wild-type burn). Additional groups of iNOS KO and wild-type mice served as appropriate sham burn groups (iNOS/KO sham and wild-type sham). Cardiac function was assessed 24 h postburn by perfusing hearts ( n = 7–10 mice/group). Burn trauma in wild-type mice impaired cardiac function as indicated by the lower left ventricular pressure (LVP, 67 ± 2 mmHg) compared with that measured in wild-type shams (94 ± 2 mmHg, P < 0.001), a lower rate of LVP rise (+dP/d tmax, 1,620 ± 94 vs. 2,240 ± 58 mmHg/s, P < 0.001), and a lower rate of LVP fall (–dP/d tmax, 1,200 ± 84 vs. 1,800 ± 42 mmHg/s, P < 0.001). Ventricular function curves confirmed significant contractile dysfunction after burn trauma in wild-type mice. Burn trauma in iNOS KO mice produced fewer cardiac derangements compared with those observed in wild-type burns (LVP: 78 ± 5 mmHg; +dP/d t: 1,889 ± 160 mmHg/s; –dP/d t: 1,480 ± 154 mmHg/s). The use of a pharmacological approach to inhibit iNOS (aminoguanidine, given ip) in additional wild-type shams and burns confirmed the iNOS KO data. Whereas the absence of iNOS attenuated burn-mediated cardiac contractile dysfunction, these experiments did not determine the contribution of cardiac-derived NO versus NO generated by immune cells. However, our data indicate a role for NO in cardiac dysfunction after major trauma.
TNF-α-dependent bilateral renal injury is induced by unilateral renal ischemia-reperfusion
Authors: Meldrum Kirstan K. , Meldrum Daniel R. , Meng Xianzhong , Ao Lihua , Harken Alden H.
Published: March 2015

While tumor necrosis factor (TNF)-α is an important mediator of renal ischemia-reperfusion (I/R) injury, its role in contralateral renal injury after isolated renal ischemia remains unknown. We therefore investigated the effect of isolated left renal ischemia on the nonischemic contralateral kidney. To study this, male Sprague-Dawley rats were anesthetized and exposed to varying degrees of left renal I/R injury. Both kidneys were subsequently harvested, serum samples were obtained, and TNF-α protein expression (ELISA), TNF-α mRNA content (RT-PCR), TNF-α immunolocalization, and neutrophil infiltration (myeloperoxidase assay) were determined. The effect of TNF-α on neutrophil infiltration was assessed by neutralizing TNF-α with TNF binding protein (TNF-BP) before left renal I/R injury. TNF-α protein expression, TNF-α mRNA induction, and neutrophil infiltration increased significantly in both kidneys after unilateral renal I/R injury. Furthermore, the administration of TNF-BP before unilateral renal I/R substantially reduced the degree of neutrophil infiltration bilaterally. These results constitute the initial demonstration that unilateral renal I/R induces bilateral TNF-α production and neutrophil infiltration through a TNF-α-dependent mechanism.
Nitric oxide-epoxygenase interactions and arachidonate-induced dilation of rat renal microvessels
Authors: Udosen I. T. , Jiang H. , Hercule H. C. , Oyekan A. O.
Published: March 2015

Nitric oxide (NO) is an inhibitor of hemoproteins including cytochrome P-450 enzymes. This study tested the hypothesis that NO inhibits cytochrome P-450 epoxygenase-dependent vascular responses in kidneys. In rat renal pressurized microvessels, arachidonic acid (AA, 0.03–1 μM) or bradykinin (BK, 0.1–3 μM) elicited NO- and prostanoid-independent vasodilation. Miconazole (1.5 μM) or 6-(2-propargyloxyphenyl)hexanoic acid (30 μM), both of which are inhibitors of epoxygenase enzymes, or the fixing of epoxide levels with 11,12-epoxyeicosatrienoic acid (11,12-EET; 1 and 3 μM) inhibited these responses. Apamin (1 μM), which is a large-conductance Ca2+-activated K+(BKCa) channel inhibitor, or 18α-glycyrrhetinic acid (30 μM), which is an inhibitor of myoendothelial gap junctional electromechanical coupling, also inhibited these responses. NO donors spermine NONOate (1 and 3 μM) or sodium nitroprusside (0.3 and 3 μM) but not 8-bromo-cGMP (100 μM), which is an analog of cGMP (the second messenger of NO), blunted the dilation produced by AA or BK in a reversible manner without affecting that produced by hydralazine. However, the non-NO donor hydralazine did not affect the dilatory effect of AA or BK. Spermine NONOate did not affect the dilation produced by 11,12-EET, NS-1619 (a BKCachannel opener), or cromakalim (an ATP-sensitive K+channel opener). AA and BK stimulated EET production, whereas hydralazine had no effect. On the other hand, spermine NONOate (3 μM) attenuated basal (19 ± 7%; P < 0.05) and AA stimulation (1 μM, 29 ± 9%; P < 0.05) of renal preglomerular vascular production of all regioisomeric EETs: 5,6-; 8,9-; 11,12-; and 14,15-EET. These results suggest that NO directly and reversibly inhibits epoxygenase-dependent dilation of rat renal microvessels without affecting the actions of epoxides on K+channels.
Echocardiographic assessment of cardiac function in diabeticdb/db and transgenic db/db-hGLUT4 mice
Authors: Semeniuk Lisa M. , Kryski Albert J. , Severson David L.
Published: March 2015

Control db/+ and diabetic db/ db mice at 6 and 12 wk of age were subjected to echocardiography to determine whether contractile function was reduced in vivo and restored in transgenic db/ db-human glucose transporter 4 (hGLUT4) mice (12 wk old) in which cardiac metabolism has been normalized. Systolic function was unchanged in 6-wk-old db/ db mice, but fractional shortening and velocity of circumferential fiber shortening were reduced in 12-wk-old db/ db mice (43.8 ± 2.1% and 8.3 ± 0.5 circs/s, respectively) relative to db/+ control mice (59.5 ± 2.3% and 11.8 ± 0.4 circs/s, respectively). Doppler flow measurements were unchanged in 6-wk-old db/ db mice. The ratio of E and A transmitral flows was reduced from 3.56 ± 0.29 in db/+ mice to 2.40 ± 0.20 in 12-wk-old db/ dbmice, indicating diastolic dysfunction. Thus a diabetic cardiomyopathy with systolic and diastolic dysfunction was evident in 12-wk-old diabetic db/ db mice. Cardiac function was normalized in transgenic db/ db-hGLUT4 mice, indicating that altered cardiac metabolism can produce contractile dysfunction in diabetic db/ db hearts.
RhoA/Rho kinase and nitric oxide modulate the agonist-induced pulmonary artery diameter response time
Authors: Boer Christa , van der Linden Peter J. W. , Scheffer Gert Jan , Westerhof Nico , de Lange Jaap J. , Sipkema Pieter
Published: March 2015

We studied the amplitude and response time (RT; time to 50% of maximal response) of pulmonary vasoreactivity and investigated whether the characteristics of pulmonary vasoreactivity could be modulated by endothelium removal, nitric oxide (NO) synthase inhibition [ N G-nitro-l-arginine (l-NNA)], RhoA activation [lysophosphatidic acid (LPA)] and Rho kinase inhibition (Y-27632). Slow acetylcholine-induced pulmonary vasodilation (262 ± 5 s) was not due to the RT of endothelial NO release (45–55 s) and was always longer than RT in renal arteries (15 ± 4 s). The rate-determining step is located in the smooth muscle cells. This was confirmed by the existing differences between the RT of the NO solution and KCl-induced renal and pulmonary vasoreactivity in endothelium-denuded arteries. We found that the pulmonary contractile amplitude increases and the RT decreases byl-NNA or LPA. In contrast, Y-27632 reduced the contractile amplitude and increased the RT in pulmonary arteries. These phenomena were dependent on the contractile stimulus (phenylephrine or KCl). In conclusion, slow pulmonary vasoreactivity is a smooth muscle cell characteristic that can be enhanced by RhoA and NO or endothelium removal. These effects were counteracted by Rho kinase inhibition. We show a role for RhoA/Rho kinase and NO in the modulation of pulmonary vascular reactivity.
Left ventricular geometry immediately following defibrillation: shock-induced relaxation
Authors: De Jongh Amy L. , Ramanathan Vijaya , Hoffmeister Brent K. , Malkin Robert A.
Published: March 2015

A previous two-dimensional (2D) ultrasound study suggested that there is relaxation of the myocardium after defibrillation. The 2D study could not measure activity occurring within the first 33 ms after the shock, a period that may be critical for discriminating between shock- and excitation-induced relaxation. The objective of our study was to determine the left ventricular (LV) geometry during the first 33 ms after defibrillation. Biphasic defibrillation shocks were delivered 5–50 s after the induction of ventricular fibrillation in each of the seven dogs. One-dimensional, short-axis ultrasound images of the LV cavity were acquired at a rate of 250 samples/s. The LV cavity diameter was computed from 32 ms before to 32 ms after the shock. Preshock and postshock percent changes in LV diameter were analyzed as a function of time with the use of regression analysis. The normalized mean pre- and postshock slopes (0.2 ± 2.2 and 3.3 ± 7.9% per 10 ms) were significantly different ( P < 0.01). The postshock slope was positive ( P < 0.005). Our results confirm that the bulk of the myocardium is relaxing immediately after defibrillation.
Contribution of endothelin to pulmonary vascular tone under normoxic and hypoxic conditions
Authors: Johnson Wendy , Nohria Anju , Garrett Leslie , Fang James C. , Igo James , Katai Miyuki , Ganz Peter , Creager Mark A.
Published: March 2015

The contribution of endothelin to resting pulmonary vascular tone and hypoxic pulmonary vasoconstriction in humans is unknown. We studied the hemodynamic effects of BQ-123, an endothelin type A receptor antagonist, on healthy volunteers exposed to normoxia and hypoxia. Hemodynamics were measured at room air and after 15 min of exposure to hypoxia (arterial Po 2 99.8 ± 1.8 and 49.4 ± 0.4 mmHg, respectively). Measurements were then repeated in the presence of BQ-123. BQ-123 decreased pulmonary vascular resistance (PVR) 26% and systemic vascular resistance (SVR) 21%, whereas it increased cardiac output (CO) 22% (all P < 0.05). Hypoxia raised CO 28% and PVR 95%, whereas it reduced SVR 23% (all P< 0.01). During BQ-123 infusion, hypoxia increased CO 29% and PVR 97% and decreased SVR 22% (all P < 0.01). The pulmonary vasoconstrictive response to hypoxia was similar in the absence and presence of BQ-123 [ P = not significant (NS)]. In vehicle-treated control subjects, hypoxic pulmonary vasoconstriction did not change with repeated exposure to hypoxia ( P = NS). Endothelin contributes to basal pulmonary and systemic vascular tone during normoxia, but does not mediate the additional pulmonary vasoconstriction induced by acute hypoxia.
High-salt diet depresses acetylcholine reactivity proximal to NOS activation in cerebral arteries
Authors: Sylvester Francis A. , Stepp David W. , Frisbee Jefferson C. , Lombard Julian H.
Published: March 2015

Rats were fed a low-salt (LS; 0.4% NaCl) or high-salt (HS; 4.0% NaCl) diet for 3 days, and the responses of isolated cerebral arteries to acetylcholine (ACh), the nitric oxide (NO)-dependent dilator bradykinin, and the NO donor 6-(2-hydroxy-1-methyl-2-nitrosohydrazino)- N-methyl-1-hex-anamine (NOC-9) were determined. ACh-induced vasodilation and NO release, assessed with the fluorescent NO indicator 4,5-diaminofluorescein (DAF-2) diacetate, were eliminated with the HS diet. Inhibition of cyclooxygenase, cytochrome P-450 epoxygenase, and acetylcholinesterase did not alter ACh responses. Bradykinin and NOC-9 caused a similar dilation in cerebral arteries of all groups. Arteries from animals on LS or HS diets exhibited similar levels of basal superoxide (O[Formula: see text]) production, assessed by dihydroethidine fluorescence, and ACh responses were unaffected by O[Formula: see text] scavengers. Muscarinic type 3 receptor expression was unaffected by dietary salt intake. These results indicate that 1) a HS diet attenuates ACh reactivity in cerebral arteries by inhibiting NO release, 2) this attenuation is not due to production of a cyclooxygenase-derived vasoconstrictor or elevated O[Formula: see text] levels, and 3) alteration(s) in ACh signaling are located upstream from NO synthase.
Effects of ACh and adenosine mediated by Kir3.1 and Kir3.4 on ferret ventricular cells
Authors: Dobrzynski H. , Janvier N. C. , Leach R. , Findlay J. B. C. , Boyett M. R.
Published: March 2015

The inotropic effects of ACh and adenosine on ferret ventricular cells were investigated with the action potential-clamp technique. Under current clamp, both agonists resulted in action potential shortening and a decrease in contraction. Under action potential clamp, both agonists failed to decrease contraction substantially. In the absence of agonist, application of the short action potential waveform (recorded previously in the presence of agonist) also resulted in a decrease in contraction. Under action potential clamp, application of ACh resulted in a Ba2+-sensitive outward current with the characteristics of muscarinic K+ current ( I K,ACh); the presence of the muscarinic K+ channel was confirmed by PCR and immunocytochemistry. In the absence of agonist, on application of the short ACh action potential waveform, the decrease in contraction was accompanied by loss of the inward Na+/Ca2+exchange current ( I NaCa). ACh also inhibited the background inward K+ current ( I K,1). It is concluded that ACh activates I K,ACh, inhibits I K,1, and indirectly inhibits I NaCa; this results in action potential shortening, decrease in contraction, and, as a result of the inhibition of I K,1, minimum decrease in excitability.
Nitric oxide-independent effects of tempol on sympathetic nerve activity and blood pressure in DOCA-salt rats
Authors: Xu Hui , Fink Gregory D. , Galligan James J.
Published: March 2015

The role of sympathetic nerves and nitric oxide (NO) in tempol-induced cardiovascular responses was evaluated in urethane-anesthetized sham and deoxycorticosterone acetate (DOCA)-salt-treated (DOCA-salt) rats. Tempol (30–300 μmol/kg iv), a superoxide (O[Formula: see text]) scavenger, decreased renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), and heart rate (HR) in DOCA-salt and sham rats. The antioxidants tiron and ascorbate did not alter MAP, HR, or RSNA in any rat. Tempol responses were unaffected after sham rats were treated with NG-nitro-l-arginine (l-NNA, 13 mg/kg). In DOCA-salt rats, l-NNA reduced tempol-induced depressor responses but not the inhibition of HR or RSNA. Tempol did not significantly decrease MAP, HR, or RSNA after hexamethonium (30 mg/kg iv) treatment in any rat. Dihydroethidine histochemistry revealed increased O[Formula: see text] levels in arteries and veins from DOCA-salt rats. Tempol treatment in vitro reduced O[Formula: see text] levels in arteries and veins from DOCA-salt rats. In conclusion, tempol-induced depressor responses are mediated largely by NO-independent sympathoinhibition in sham and DOCA-salt rats. There is an additional interaction between NO and tempol that contributes to depressor responses in DOCA-salt rats.
Vascular and metabolic response to isolated small muscle mass exercise: effect of age
Authors: Lawrenson L. , Poole J. G. , Kim J. , Brown C. , Patel P. , Richardson R. S.
Published: March 2015

To determine the effect of age on quadriceps muscle blood flow (QMBF), leg vascular resistance (LVR), and maximum oxygen uptake (QV̇O2 max), a thermal dilution technique was used in conjunction with arterial and venous femoral blood sampling in six sedentary young (19.8 ± 1.3 yr) and six sedentary old (66.5 ± 2.1 yr) males during incremental knee extensor exercise (KE). Young and old attained a similar maximal KE work rate (WRmax) (young: 25.2 ± 2.1 and old: 24.1 ± 4 W) and QV̇O2 max (young: 0.52 ± 0.03 and old: 0.42 ± 0.05 l/min). QMBF during KE was lower in old subjects by ∼500 ml/min across all work rates, with old subjects demonstrating a significantly lower QMBF/W (old: 174 ± 20 and young: 239 ± 46 ml · min–1 · W–1). Although the vasodilatory response to incremental KE was ∼142% greater in the old (young: 0.0019 and old: 0.0046 mmHg · min · ml–1 · W–1), consistently elevated leg vascular resistance (LVR) in the old, ∼80% higher LVR in the old at 50% WR and ∼40% higher LVR in the old at WRmax (young: 44.1 ± 3.6 and old: 31.0 ± 1.7 mmHg · min · ml–1), dictated that during incremental KE the LVR of the old subjects was never less than that of the young subjects. Pulse pressures, indicative of arterial vessel compliance, were ∼36% higher in the old subjects across all work rates. In conclusion, well-matched sedentary young and old subjects with similar quadriceps muscle mass achieved a similar WRmax and QV̇O2 max during incremental KE. The old subjects, despite a reduced QMBF, had a greater vasodilatory response to incremental KE. Given that small muscle mass exercise, such as KE, utilizes only a fraction of maximal cardiac output, peripheral mechanisms such as consistently elevated leg vascular resistance and greater pulse pressures appear to be responsible for reduced blood flow persisting throughout graded KE in the old subjects.
Vascular endothelial growth factor mRNA expression and arteriovenous balance in response to prolonged, submaximal exercise in humans
Authors: Hiscock N. , Fischer C. P. , Pilegaard H. , Pedersen B. K.
Published: March 2015

Angiogenesis, the growth of new blood vessels from existing ones, occurs in the skeletal muscle as an adaptive response to exercise that satisfies the increased requirement of this tissue for oxygen delivery and metabolic processes. Of the factors that have been identified to regulate this process, the endothelial cell mitogen vascular endothelial growth factor (VEGF) has been proposed to play a key role. The aim of this study was to measure the skeletal muscle VEGF mRNA content and arteriovenous protein balance across the working leg in response to a single bout of prolonged, submaximal exercise. Seven physically active males completed 3 h of two-legged kicking ergometry. Muscle biopsies were collected from the vastus lateralis muscle from both working legs, and blood samples were collected from one femoral artery and femoral vein before, during, and in recovery from exercise. We show that the exercise stimulus elicited a decrease in VEGF protein arteriovenous balance across the exercising leg ( P = 0.007), and a ninefold elevation in skeletal muscle VEGF mRNA expression ( P < 0.001). The changes in VEGF protein balance and mRNA content were most pronounced 1 h after the cessation of exercise. In conclusion, these findings demonstrate that submaximal exercise, suitable for humans with low CV fitness, induces a decrease in VEGF arteriovenous balance that is likely to be of clinical significance in promoting angiogenic effects.
Muscle metaboreflex control of coronary blood flow
Authors: Ansorge Eric J. , Shah Sachin H. , Augustyniak Robert A. , Rossi Noreen F. , Collins Heidi L. , O'Leary Donal S.
Published: March 2015

We investigated the effect of muscle metaboreflex activation on left circumflex coronary blood flow (CBF) and vascular conductance (CVC) in conscious, chronically instrumented dogs during treadmill exercise ranging from mild to severe workloads. Metaboreflex responses were also observed during mild exercise with constant heart rate (HR) of 225 beats/min and β1-adrenergic receptor blockade to attenuate the substantial reflex increases in cardiac work. The muscle metaboreflex was activated via graded partial occlusion of hindlimb blood flow. During mild exercise, with muscle metaboreflex activation, hindlimb ischemia elicited significant reflex increases in mean arterial pressure (MAP), HR, and cardiac output (CO) (+39.0 ± 5.2 mmHg, +29.9 ± 7.7 beats/min, and +2.0 ± 0.4 l/min, respectively; all changes, P < 0.05). CBF increased from 51.9 ± 4.3 to 88.5 ± 6.6 ml/min, ( P < 0.05), whereas no significant change in CVC occurred (0.56 ± 0.06 vs. 0.59 ± 0.05 ml · min−1 · mmHg−1; P > 0.05). Similar responses were observed during moderate exercise. In contrast, with metaboreflex activation during severe exercise, no further increases in CO or HR occurred, the increases in MAP and CBF were attenuated, and a significant reduction in CVC was observed (1.00 ± 0.12 vs. 0.90 ± 0.13 ml · min−1 · mmHg−1; P < 0.05). Similarly, when the metaboreflex was activated during mild exercise with the rise in cardiac work lessened (via constant HR and β1-blockade), no increase in CO occurred, the MAP and CBF responses were attenuated (+15.6 ± 4.5 mmHg, +8.3 ± 2 ml/min), and CVC significantly decreased from 0.63 ± 0.11 to 0.53 ± 0.10 ml · min−1 · mmHg−1. We conclude that the muscle metaboreflex induced increases in sympathetic nerve activity to the heart functionally vasoconstricts the coronary vasculature.
α2-Adrenergic stimulation is protective against ischemia-reperfusion-induced ventricular arrhythmias in vivo
Authors: Cai John J. , Morgan Donald A. , Haynes William G. , Martins James B. , Lee Hon-Chi
Published: March 2015

We previously reported that α2-adrenergic receptor (α2-AR) stimulation in Purkinje fibers in vitro prolongs action potential duration and suppresses β-adrenergic-induced delayed afterdepolarizations and sustained triggered activities. We examined the effects of α2-AR stimulation on reperfusion-induced ventricular arrhythmias [ventricular tachycardia/ventricular fibrillation (VT/VF)] in vivo. Arterial blood pressure, heart rate, surface electrocardiogram, and renal sympathetic nerve activities were recorded simultaneously in Sprague-Dawley rats. The incidence of VT/VF was 87.5% for controls, 50% for the β-blocker group, 72% for the α1-blocker group, and 12.5% for the α1 + β-blockers group (unopposed α2-adrenergic activation). Direct α2-AR stimulation with UK-14304 also prevented VT/VF. These effects were reversed by the α2-adrenergic antagonist yohimbine. Increases in renal sympathetic nerve activity were associated with left anterior descending coronary artery ligation and reperfusion (33 ± 1.5 and 62 ± 1.7% over baseline, respectively) in controls. Similar patterns were observed among all experimental groups irrespective of the incidence of VT/VF on reperfusion. We conclude that α2-AR stimulation has a potent antiarrhythmic effect on ischemia-reperfusion-induced VT/VF in vivo and that this effect is not centrally mediated.
Regulation of the S100B gene by α1-adrenergic stimulation in cardiac myocytes
Authors: Tsoporis James N. , Marks Alexander , Van Eldik Linda J. , O'Hanlon David , Parker Thomas G.
Published: March 2015

We previously reported that S100B, a 20-kDa Ca2+-binding homodimer, inhibited the postinfarct myocardial hypertrophic response mediated by α1-adrenergic stimulation through the protein kinase C (PKC) signaling pathway. In the present study, we examined whether the same pathway induced the S100B gene, supporting the hypothesis that S100B is a feedback negative regulator of this pathway. We transfected cultured neonatal rat cardiac myocytes with a luciferase reporter gene driven by the maximal human S100B promoter and progressively shorter segments of this promoter sequentially deleted from the 5′ end. We identified a basic promoter essential for transcription spanning 162 bp upstream of the transcription initiation site and positive (at −782/−162 and −6,689/−4,463) and negative (at −4,463/−782) myocyte-selective regulatory elements. We showed that the basic and maximal S100B promoters were activated specifically by α1-adrenergic agonists through the α1A-adrenergic receptor, but not by any other trophic hormonal stimuli. The activation of the S100B promoter was mediated through the PKC signaling pathway. Transcription enhancer factor-1 (TEF-1) and related to TEF-1 (RTEF-1) influenced transcription from the maximal, but not the basic, promoter implicating active MCAT elements upstream from the basic promoter. Acting in opposing fashions, TEF-1 transrepressed the S100B promoter and RTEF-1 transactivated the promoter. Our results suggest that α1-adrenergic stimulation induces the S100B gene after myocardial infarction through the PKC signaling pathway and that this induction is modulated by TEF-1 and RTEF-1.
Depressed transient outward potassium current density in catecholamine-depleted rat ventricular myocytes
Authors: Bru-Mercier Gilles , Deroubaix Edith , Rousseau Delphine , Coulombe Alain , Renaud Jean-François
Published: March 2015

The effect of catecholamine depletion (induced by prior treatment with reserpine) was studied in Wistar rat ventricular myocytes using whole cell voltage-clamp methods. Two calcium-independent outward currents, the transient outward potassium current ( I to) and the sustained outward potassium current ( I sus), were measured. Reserpine treatment decreased tissue norepinephrine content by 97%. Action potential duration in the isolated perfused heart was significantly increased in reserpine-treated hearts. In isolated ventricular myocytes, I to density was decreased by 49% in reserpine-treated rats. This treatment had no effect on I sus. The I tosteady-state inactivation-voltage relationship and recovery from inactivation remained unchanged, whereas the conductance-voltage activation curve for reserpine-treated rats was significantly shifted (6.7 mV) toward negative potentials. The incubation of myocytes with 10 μM norepinephrine for 7–10 h restored I to, an effect that was abolished by the presence of actinomycin D. Norepinephrine (0.5 μM) had no effect on I to. However, in the presence of both 0.5 μM norepinephrine and neuropeptide Y (0.1 μM), I to density was restored to its control value. These results suggest that the sympathetic nervous system is involved in I to regulation. Sympathetic norepinephrine depletion decreased the number of functional channels via an effect on the α-adrenergic cascade and norepinephrine is able to restore expression of I to channels.
A new inbred Wistar-Kyoto rat substrain exhibiting apparent salt sensitivity and borderline hypertension
Authors: Alemayehu Adamu , Breen Laura , Printz Morton P.
Published: March 2015

The normotensive Wistar-Kyoto (WKY) rat strain is a traditional control for the spontaneously hypertensive rat (SHR). We found trait differences between two inbred normotensive WKY strains, derived originally from different vendors, and compared these two strains from La Jolla-Taconic Farms (WKY/lj-tf) and La Jolla-Charles River (WKY/lj-cr) with the inbred SHR/lj-cr for cardiovascular, diurnal, and activity traits under normal and high (8%) NaCl diets. Marked genetic diversity was found between the two vendor-derived WKY. By using an extended study design and radiotelemetry, we compared WKY/lj-cr, WKY/lj-tf, and SHR/lj-cr with the following results: systolic pressure (120 ± 1, 133 ± 1, 168 ± 3 mmHg, respectively); diurnal variation in heart rate (ΔHR: 46 ± 3, 71 ± 4, 57 ± 2 beats/min, respectively); and salt sensitivity of arterial pressure (Δsystolic: 10 ± 1, 21 ± 1, 20 ± 1 mmHg, respectively). The WKY/lj-tf genotype apparently results in compromised control of arterial pressure and heart rate, especially during high NaCl intake, and greater susceptibility to high pressure (i.e., high NaCl-induced secondary changes). WKY/lj-tf thus constitutes a new inbred borderline hypertensive WKY substrain offering unique opportunities for genomic studies into the development of genetic hypertension.
Monocyte chemotactic protein-1 directly induces human vascular smooth muscle proliferation
Authors: Selzman Craig H. , Miller Stephanie A. , Zimmerman Michael A. , Gamboni-Robertson Fabia , Harken Alden H. , Banerjee Anirban
Published: March 2015

Although monocyte chemotactic protein-1 (MCP-1) is best known for its ability to recruit mononuclear cells, few studies have examined the effects of this chemokine on other events in the vascular response to injury. The purpose of the present study was to determine the influence of MCP-1 on human vascular smooth muscle (VSMC) proliferation. MCP-1 induced concentration-dependent VSMC proliferation as measured by bromodeoxyuridine (BrdU) uptake. Direct cell counting demonstrated a twofold increase in VSMC after stimulation with MCP-1. This mitogenic effect was similar to that observed with the prototypical atherogenic cytokine platelet-derived growth factor. Immunohistochemistry and Western blot analysis revealed that MCP-1 increased both proliferating nuclear cell antigen and cyclin A expression. Whereas MCP-1 did not promote nuclear factor-κB activation, MCP-1-induced VSMC proliferation appeared to be dependent on phosphotidylinositol 3-kinase activation. In conclusion, MCP-1 directly induces VSMC growth, which is associated with activation of cell cycle proteins and intracellular proliferative signals. Within the inflammatory paradigm of vascular remodeling, these data suggest that MCP-1 is more than simply a chemokine but also a potent mitogen for VSMC proliferation.