bluuebunny/crossref_metadata_2025 huggingface dataset:
Cotransmission from sympathetic vasoconstrictor neurons to small cutaneous arteries in vivo
Authors: Morris Judy L.
Published: December 2017

This study has characterized constrictions of small cutaneous arteries in the guinea pig ear in response to electrical stimulation of the cervical sympathetic nerve (SNS) in vivo. Video microscopy and on-line image analysis were used to examine diameter changes of ear arteries (80–140 μm resting diameter) in anesthetized guinea pigs. Trains of 50–300 impulses, but not single pulses or short trains, produced frequency-dependent (2–20 Hz) constrictions. The purinoceptor antagonist suramin (30 μM) greatly reduced constrictions produced by exogenous ATP but did not affect constrictions produced by SNS at 10 Hz or exogenous norepinephrine. The α2-adrenoceptor antagonist yohimbine (1 μM) enhanced the peak amplitude of sympathetic constrictions at lower stimulation frequencies (1–5 Hz). The amplitude of constrictions to SNS at 10 Hz was reduced, and the latency of constrictions was increased by the α1-adrenoceptor antagonist prazosin (1 μM). Constrictions to SNS at 10 Hz remaining after prazosin treatment were reduced in amplitude by dihydroergotamine (2 μM) and were attenuated further by the neuropeptide Y Y1-receptor antagonist 1229U91 (0.3 μM). Thus norepinephrine and neuropeptide Y act as cotransmitters to mediate sympathetic constriction of small ear arteries at higher stimulation frequencies (10 Hz), but ATP does not seem to contribute directly to these constrictions.
Energy expenditure by Ba2+contracture in rat ventricular slices derives from cross-bridge cycling
Authors: Kohzuki Hisaharu , Misawa Hiromi , Sakata Susumu , Ohga Yoshimi , Suga Hiroyuki , Takaki Miyako
Published: December 2017

To clarify the energy-expenditure mechanism during Ba2+contracture of mechanically unloaded rat left ventricular (LV) slices, we measured myocardial O2consumption (V˙o2) of quiescent slices in Ca2+-free Tyrode solution andV˙o2during Ba2+contracture by substituting Ca2+with Ba2+. We then investigated the effects of cyclopiazonic acid (CPA) and 2,3-butanedione monoxime (BDM) on the Ba2+contractureV˙o2. The Ca2+-freeV˙o2corresponds to that of basal metabolism (2.32 ± 0.53 ml O2⋅ min−1⋅ 100 g LV−1). Ba2+increased theV˙o2in a dose-dependent manner (from 0.3 to 3.0 mmol/l) from 110 to 150% of basal metabolic V˙o2. Blockade of the sarcoplasmic reticulum (SR) Ca2+pump by CPA (10 μmol/l) did not at all decrease the Ba2+-activatedV˙o2. BDM (5 mmol/l), which specifically inhibits cross-bridge cycling, reduced the Ba2+activatedV˙o2almost to basal metabolic V˙o2. These energetic results revealed that the Ba2+-activatedV˙o2was used for the cross-bridge cycling but not for the Ca2+handling by the SR Ca2+pump.
Ventricular response in atrial fibrillation: random or deterministic?
Authors: Stein Kenneth M. , Walden Jeff , Lippman Neal , Lerman Bruce B.
Published: December 2017

The ventricular response in atrial fibrillation is often described as “chaotic,” but this has not been demonstrated in the strict mathematical sense. A defining feature of chaotic systems is sensitive dependence on initial conditions: similar sequences evolve similarly in the short term but then diverge exponentially. We developed a nonlinear predictive forecasting algorithm to search for predictability and sensitive dependence on initial conditions in the ventricular response during atrial fibrillation. The algorithm was tested for simulated R-R intervals from a linear oscillator with and without superimposed white noise, a chaotic signal (the logistic map) with and without superimposed white noise, and a pseudorandom signal and was then applied to R-R intervals from 16 chronic atrial fibrillation patients. Short-term predictability was demonstrated for the linear oscillators, without loss of predictive ability farther into the future. The chaotic system demonstrated high short-term predictability that declined rapidly further into the future. The pseudorandom signal was unpredictable. The ventricular response in atrial fibrillation was weakly predictable (statistically significant predictability in 8 of 16 patients), without sensitive dependence on initial conditions. Although the R-R interval sequence is not completely unpredictable, a low-dimensional chaotic attractor does not govern the irregular ventricular response during atrial fibrillation.
Effects of endothelin on spontaneous contractions in lymph vessels
Authors: Sakai Hiroshi , Ikomi Fumitaka , Ohhashi Toshio
Published: December 2017

A mode of action of endothelin (ET) on spontaneous contractions was investigated in ring preparations of isolated bovine mesenteric lymphatics. ET-1 at concentrations between 10−10and 10−9M caused a dose-dependent increase in the frequency of spontaneous contractions. The specific ETA-receptor antagonist BQ-123 (5 × 10−7M) caused a significant inhibition of the ET-1-induced positive chronotropic effect in the ring preparations with and without the endothelium. Mechanical denudation of the lymphatic endothelial cells produced a significant potentiation of the ET-induced positive chronotropic effect. BQ-3020 (10−8–10−7M), a selective ETB-receptor agonist, induced dose dependently negative chronotropic and inotropic effects on the spontaneous contractions in the ring preparations with intact endothelium. Mechanical removal of the endothelium caused a significant reduction of the BQ-3020-induced negative chronotropic and inotropic effects. The ET-1-induced positive chronotropic effect was potentiated by pretreatment with Nω-nitro-l-arginine methyl ester (l-NAME) (10−5M) but unaffected by aspirin (10−5M). Additional treatment with l-arginine (10−4M) completely reversed the l-NAME-mediated potentiation of the ET-induced chronotropic effect. These results suggest that stimulation of ETAreceptors on the lymphatic smooth muscles causes a positive chronotropic effect on the spontaneous contractions, and stimulation of ETBreceptors on the lymphatic endothelial cells induces a release of nitric oxide, which results in the chronotropic and inotropic effects on spontaneous contractions in isolated bovine mesenteric lymphatics.
Role of K ATP + channels and adenosine in regulation of coronary blood flow in the hypertrophied left ventricle
Authors: Melchert Peter J. , Duncker Dirk J. , Traverse Jay H. , Bache Robert J.
Published: December 2017

In the hypertrophied heart, increased extravascular forces acting to compress the intramural coronary vessels might require augmentation of metabolic vasodilator mechanisms to maintain adequate coronary blood flow. Vascular smooth muscle ATP-sensitive potassium ([Formula: see text]) channel activity is important in metabolic coronary vasodilation, and adenosine contributes to resistance vessel dilation in the hypoperfused heart. Consequently, this study was performed to determine whether[Formula: see text] channels and adenosine have increased importance in exercise-induced coronary vasodilation in the hypertrophied left ventricle. Studies were performed in dogs in which banding of the ascending aorta had resulted in a 66% increase in left ventricular mass in comparison with historic normal animals. Treadmill exercise resulted in increases of coronary blood flow that were linearly related to the increase of heart rate or rate-pressure product. During resting conditions, [Formula: see text]channel blockade with glibenclamide caused a 17 ± 5% decrease in coronary blood flow, similar to that previously observed in normal hearts. Unlike normal hearts, however, glibenclamide blunted the increase in coronary flow that occurred during exercise, causing a significant decrease in the slope of the relationship between coronary flow and the rate-pressure product. Adenosine receptor blockade with 8-phenyltheophylline did not alter coronary blood flow at rest or during exercise. Furthermore, even after[Formula: see text] channel blockade with glibenclamide, the addition of 8-phenyltheophylline had no effect on coronary blood flow. This finding was different from normal hearts, in which the addition of adenosine receptor blockade after glibenclamide impaired exercise-induced coronary vasodilation. The data suggest that, in comparison with normal hearts, hypertrophied hearts have increased reliance on opening of [Formula: see text] channels to augment coronary flow during exercise. Contrary to the initial hypothesis, however, adenosine was not mandatory for exercise-induced coronary vasodilation in the hypertrophied hearts either during control conditions or when [Formula: see text] channel activity was blocked with glibenclamide.
Opioid modulation of calcium current in cultured sensory neurons: μ-modulation of baroreceptor input
Authors: Hamra Mary , McNeil Robert S. , Runciman Martin , Kunze Diana L.
Published: December 2017

We used the whole cell open-patch or perforated-patch technique to characterize μ-opioid modulation of Ca2+ current ( I Ca) in nodose sensory neurons and in a specific subpopulation of nodose cells, aortic baroreceptor neurons. The μ-opiate receptor agonist Tyr-d-Ala-Gly-MePhe-Gly-ol enkephalin (DAGO) inhibited I Ca in 95% of neonatal [postnatal day (P)1–P3] nodose neurons. To the contrary, only 64% of juvenile cells (P20–P35) and 61% of adult cells (P60–P110) responded to DAGO. DAGO-mediated inhibition of I Ca was naloxone sensitive, irreversible in the presence of guanosine 5′- O-(3-thiotriphosphate), absent with guanosine 5′- O-(2-thiodiphosphate), and eliminated with pertussis toxin; DAGO’s inhibition of I Ca was G protein mediated. Incubation of neurons with ω-conotoxin GVIA eliminated the effect of DAGO in neonatal but not in juvenile cells. In the latter, DAGO reduced 37% of the current remaining in the presence of ω-conotoxin. In the subset of nodose neurons, aortic baroafferents, the effect of DAGO was concentration dependent, with an IC50 of 1.82 × 10−8 M. DAGO slowed activation of I Ca, but activation curves constructed from tail currents were the same with and without DAGO (100 nM). In summary, μ-opiate modulation of I Ca in nodose neurons was demonstrated in three age groups, including specifically labeled baroafferents. The demonstration of a mechanism of action of μ-opioids on baroreceptor afferents provides a basis for the attenuation of the baroreflex that occurs at the level of the nucleus tractus solitarii.
Effects of long-term, high-altitude hypoxia on the capillarity of the ovine fetal heart
Authors: Lewis A. M. , Mathieu-Costello O. , McMillan P. J. , Gilbert R. D.
Published: December 2017

To determine the effect of chronic hypoxia on myocardial capillarity, we exposed pregnant ewes to an altitude of 3,820 m from day 30 to day 139 of gestation and compared the fetus to low-altitude (∼300 m) controls. We hypothesized that capillarity would increase in the hypoxic myocardium to optimize oxygen and metabolite flux to hypoxic tissues. Fetal hearts were fixed by retrograde aortic perfusion and processed for microscopy and stereological evaluation. Fiber cross-sectional area and capillary density were measured and standardized to sarcomere length. Capillary volume density and capillary diameter were measured, capillary-to-fiber ratio and capillary length density were calculated, and the capillary anisotropy coefficient was obtained from a table of known values. Capillary-to-fiber ratio, capillary volume density, and the capillary anisotropy coefficient were not different between hypoxia and control groups. Capillary diameter was significantly larger in the right compared with the left ventricle of hypoxic but not control hearts; fiber cross-sectional area tended to be larger in the right ventricle of both groups, but this was not significant. As a result of larger fiber size, capillary density and capillary length density were significantly smaller in the right ventricle of hypoxic but not control fetal hearts. Contrary to our hypothesis, the ovine fetus does not show morphological adaptation in the myocardium after ∼109 days of high-altitude hypoxic stress.
Codistribution of NOS and caveolin throughout peripheral vasculature and skeletal muscle of hamsters
Authors: Segal Steven S. , Brett Suzanne E. , Sessa William C.
Published: December 2017

In isolated cell systems, nitric oxide synthase (NOS) activity is regulated by caveolin (CAV), a resident caveolae coat protein. Because little is known of this interaction in vivo, we tested whether NOS and caveolin are distributed together in the intact organism. Using immunohistochemistry, we investigated the localization of constitutive neuronal (nNOS) and endothelial (eNOS) enzyme isoforms along with caveolin-1 (CAV-1) and caveolin-3 (CAV-3) throughout the systemic vasculature and peripheral tissues of the hamster. The carotid artery, abdominal aorta, vena cava, femoral artery and vein, feed artery and collecting vein of the cheek pouch retractor muscle, capillaries and muscle fibers of retractor and cremaster muscles, and arterioles and venules of the cheek pouch were studied. In endothelial cells, eNOS and CAV-1 were present throughout the vasculature, whereas nNOS and CAV-3 were absent except in capillaries, which reacted for nNOS. In smooth muscle cells, nNOS and CAV-1 were also expressed systemically, whereas eNOS was absent; CAV-3 was present in the arterial but not the venous vasculature. Both nNOS and CAV-3 were located at the sarcolemma of skeletal muscle fibers, which were devoid of eNOS and CAV-1. These immunolabeling patterns suggest functional interactions between eNOS and CAV-1 throughout the endothelium, regional differences in the modulation of nNOS by caveolin isoforms in vascular smooth muscle, and modulation of nNOS by CAV-3 in skeletal muscle.
In vivo location and mechanism of EDHF-mediated vasodilation in canine coronary microcirculation
Authors: Nishikawa Yasuhiro , Stepp David W. , Chilian William M.
Published: December 2017

Responses of epicardial coronary arterioles to ACh were measured using stroboscopic fluorescence microangiography in dogs ( n = 38). ACh (0.1 and 0.5 μg ⋅ kg−1 ⋅ min−1ic) dilated small (<100 μm, 11 ± 2 and 19 ± 2%, respectively) and large (>100 μm, 6 ± 3 and 13 ± 3%, respectively) arterioles at baseline. Combined administration of N ω-monomethyl-l-arginine (l-NMMA; 1.0 μmol/min ic) and indomethacin (10 mg/kg iv) eliminated ACh-induced dilation in large coronary arterioles but only partially attenuated that in small arterioles. Suffusion of a buffer containing 60 mM KCl (high KCl) completely abolished cromakalim-induced dilation in arterioles and in combination with l-NMMA plus indomethacin completely blocked ACh-induced dilation in small arterioles. This indicated that the vasodilation to ACh that persists in small arterioles after administration of l-NMMA and indomethacin is mediated via a hyperpolarizing factor. The ACh-induced vasodilation remaining after l-NMMA and indomethacin was completely blocked by the large-conductance potassium-channel antagonist iberiotoxin or by epicardial suffusion of miconazole or metyrapone, inhibitors of cytochrome P-450 enzymes. These observations are consistent with the view that endothelium-derived hyperpolarizing factor (EDHF) is a product of cytochrome P-450 enzymes and produces vasodilation by the opening of large-conductance potassium channels. We conclude that ACh-induced dilation in large coronary arterioles is mediated mainly by nitric oxide (NO), whereas, in small arterioles both NO and EDHF mediate dilation to ACh. These data provide the first direct evidence for an in vivo role of EDHF in small coronary arterioles.
Nitroglycerin dilates coronary collateral vessels during exercise after blockade of endogenous NO production
Authors: Klassen Christopher L. , Traverse Jay H. , Bache Robert J.
Published: December 2017

In a previous study nitroglycerin failed to dilate coronary collateral vessels during exercise. This study tested the hypothesis that failure of nitroglycerin to increase collateral flow occurred because endogenous nitric oxide (NO) had activated the guanylate cyclase vasodilator pathway so that additional NO from nitroglycerin could have no additional effect. Six dogs were collateralized using intermittent 2-min occlusions of the left anterior descending coronary artery followed by permanent occlusion. One week after permanent coronary occlusion, dogs were exercised on a treadmill (heart rate 202 ± 5 beats/min), while blood flow was measured with radioactive microspheres. Blood flow to the collateral zone during control exercise was 1.90 ± 0.11 ml ⋅ min−1 ⋅ g−1compared with 2.28 ± 0.15 ml ⋅ min−1 ⋅ g−1in the normal zone ( P < 0.05); systolic wall thickening was 23 ± 3% in the collateral zone compared with 27 ± 2% in the normal zone. When N G-nitro-l-arginine (l-NNA; 20 mg/kg iv) was administered to block NO production, collateral zone flow during exercise decreased to 1.43 ± 0.20 ml ⋅ min−1 ⋅ g−1( P < 0.05), and systolic wall thickening decreased to 12 ± 4% ( P < 0.05). A subsequent infusion of nitroglycerin (2 μg ⋅ kg−1 ⋅ min−1iv) increased collateral zone blood flow to 1.65 ± 0.16 ml ⋅ min−1 ⋅ g−1( P < 0.05) and increased systolic wall thickening to 22 ± 5% ( P < 0.05). These findings demonstrate that endogenous NO contributes to collateral zone blood flow during exercise. If endogenous NO synthesis is blocked, then nitroglycerin is effective in improving collateral zone blood flow and contractile function during exercise.
Phosphorylation events associated with cyclic nucleotidedependent inhibition of smooth muscle contraction
Authors: Woodrum David A. , Brophy Colleen M. , Wingard Christopher J. , Beall Arthur , Rasmussen Howard
Published: December 2017

Activation of cyclic nucleotide-dependent signaling pathways leads to relaxation of bovine carotid artery smooth muscle contractions and is associated with increased phosphorylation of the small heat shock-related protein (HSP20). Previous reports have shown that human umbilical artery smooth muscle is uniquely resistant to cyclic nucleotide-dependent relaxation, and HSP20 is not phosphorylated. In this investigation, we determined the phosphorylation events associated with cyclic nucleotide-dependent inhibition of smooth muscle contraction. In carotid artery, activation of cyclic nucleotide-dependent signaling pathways inhibited contractile responses to serotonin but did not inhibit myosin light chain phosphorylation or oxygen consumption. The inhibition of contraction was associated with increases in HSP20 phosphorylation. In umbilical artery, activation of cyclic nucleotide-dependent signaling pathways did not inhibit serotonin-induced contraction or myosin light chain phosphorylation. The lack of contractile inhibition in umbilical artery was not associated with significant increases in HSP20 phosphorylation. In conclusion, cyclic nucleotide-dependent contractile inhibition is independent of the inhibition of myosin light chain phosphorylation or oxygen consumption but does correlate with increased HSP20 phosphorylation.
[Ca2+]ihomeostasis and cyclic nucleotide relaxation in aorta of phospholamban-deficient mice
Authors: Lalli M. Jane , Shimizu Shunichi , Sutliff Roy L. , Kranias Evangelia G. , Paul Richard J.
Published: December 2017

Phospholamban (PLB), a protein localized in the sarcoplasmic reticulum (SR), inhibits the SR Ca2+-ATPase; phosphorylation of PLB relieves this inhibition. We previously reported significant differences in contractility in aorta from mice in which the gene for PLB was ablated (PLB−). In this study, we measured intracellular Ca2+concentration ([Ca2+]i) with fura 2 in the intact mouse aorta to more directly test the hypothesis that these changes are ascribable to altered SR function in vivo. Ten micromoles per liter of the α-agonist phenylephrine (PE) increased [Ca2+]imonotonically to a steady state in the wild-type aorta. In contrast, in PLB− aorta there was an initial rapid increase to a peak [Ca2+]i, which then decreased to a steady state that was lower than that in the wild type. Upon removal of the stimulus (either PE or KCl), the decrease in [Ca2+]iwas two times as fast in the PLB− as in the wild-type aorta. There were no significant differences between PLB− and wild-type aortas in the concentration vs. force relations or the time courses of relaxation in response to forskolin or sodium nitroprusside. Interestingly, stimulation of the cAMP pathway before cGMP pathway activation resulted in a significant increase in sensitivity and a difference in relaxation parameters between PLB− and wild-type aortas. Western blot analysis indicated that the PLB-to-sarcoendoplasmic reticulum Ca2+ATPase ratio in the mouse aorta was similar to that in the heart; 20-fold more aortic than heart homogenate was required to achieve a similar level of immunoreactivity. Our data indicate that PLB can play a major role in modulating smooth muscle [Ca2+]ibut only a minor role, if any, in cyclic nucleotide-mediated relaxation.
Modulation of Na+/H+exchange isoform 1 mRNA expression in isolated rat hearts
Authors: Gan Xiaohong Tracey , Chakrabarti Subrata , Karmazyn Morris
Published: December 2017

Na+/H+exchange (NHE) has been demonstrated to mediate myocardial ischemia and reperfusion injury as well as injury produced by hydrogen peroxide (H2O2) or lysophosphatidylcholine (LPC). However, changes in gene expression in response to injurious factors have not been extensively studied. We examined Na+/H+exchange isoform 1 (NHE-1) expression using Southern detection of the RT-PCR product in response to 30 min of global ischemia with or without reperfusion in isolated rat hearts or to 30 min of exposure to either H2O2(100 μM) or LPC (5 μM). We also determined whether ischemic preconditioning (2× 5-min ischemia) alters basal NHE-1 expression or the subsequent response to insult. Ischemia with or without reperfusion increased NHE-1 expression approximately sevenfold ( P < 0.05), whereas either H2O2or LPC increased expression approximately twofold. Preconditioning reduced NHE-1 message by ∼70% ( P < 0.05) and significantly attenuated the effects of ischemia, H2O2, or LPC. The internal standard, β-globin was unaffected by any treatment. Our results indicate that NHE-1 expression is rapidly increased in response to ischemia with or without reperfusion as well as in response to H2O2or LPC. In contrast, preconditioning was associated with downregulation of NHE-1. These results may be important in furthering our understanding of NHE-1 in cardiac disease states and suggest that the antiporter adapts rapidly to cardiac conditions associated with pathology.
Chelerythrine increases Na-K-ATPase activity and limits ischemic injury in isolated rat hearts
Authors: Lundmark Jennifer L. , Ramasamy Ravichandran , Vulliet P. Richard , Schaefer Saul
Published: December 2017

Myocardial ischemia results in an increase in intracellular sodium concentration ([Na]i), which may lead to cellular injury via cellular swelling and calcium overload. Because protein kinase C (PKC) has been shown to reduce Na-K-ATPase activity, we postulated that pharmacological inhibition of PKC would directly increase Na-K-ATPase activity, reduce [Na]iduring ischemia, and provide protection from ischemic injury. Isolated rat hearts were subjected to 30 min of global ischemia with and without the specific PKC inhibitor chelerythrine. Intracellular pH, ATP, and [Na]iwere assessed using31P and23Na NMR spectroscopy, whereas Na-K-ATPase and PKC activity were determined using biochemical assays. Na/H exchanger activity was determined using the ammonium prepulse technique under nonischemic conditions. Chelerythrine increased Na-K-ATPase activity (13.76 ± 0.89 vs. 10.89 ± 0.80 mg ADP ⋅ h−1⋅ mg protein−1; P = 0.01), reduced PKC activity in both the membrane and cytosolic fractions (39% and 28% of control, respectively), and reduced creatine kinase release on reperfusion (48 ± 5 IU/g dry wt vs. 689 ± 63 IU/g dry wt; P = 0.008). The rise in [Na]iduring ischemia was significantly reduced in hearts treated with chelerythrine (peak [Na]ichelerythrine: 21.5 ± 1.2 mM; control: 31.9 ± 1.2 mM; P < 0.0001), without an effect on either acidosis (nadir pH 6.16 ± 0.05 for chelerythrine vs. 6.08 ± 0.04 for control), the rate of ATP depletion or Na/H exchanger activity. These data support the hypothesis that pharmacological inhibition of PKC before ischemia induces cardioprotection by reducing intracellular sodium overload via an increase in Na-K-ATPase activity.
Evidence for an interaction between adducin and Na+-K+-ATPase: relation to genetic hypertension
Authors: Ferrandi Mara , Salardi Sergio , Tripodi Grazia , Barassi Paolo , Rivera Rodolfo , Manunta Paolo , Goldshleger Rivka , Ferrari Patrizia , Bianchi Giuseppe , Karlish Steven J. D.
Published: December 2017

Adducin point mutations are associated with genetic hypertension in Milan hypertensive strain (MHS) rats and in humans. In transfected cells, adducin affects actin cytoskeleton organization and increases the Na+-K+-pump rate. The present study has investigated whether rat and human adducin polymorphisms differently modulate rat renal Na+-K+-ATPase in vitro. We report the following. 1) Both rat and human adducins stimulate Na+-K+-ATPase activity, with apparent affinity in tens of nanomolar concentrations. 2) MHS and Milan normotensive strain (MNS) adducins raise the apparent ATP affinity for Na+-K+-ATPase. 3) The mechanism of action of adducin appears to involve a selective acceleration of the rate of the conformational change E2(K) → E1(Na) or E2(K) ⋅ ATP → E1Na ⋅ ATP. 4) Apparent affinities for mutant rat and human adducins are significantly higher than those for wild types. 5) Recombinant human α- and β-adducins stimulate Na+-K+-ATPase activity, as do the COOH-terminal tails, and the mutant proteins display higher affinities than the wild types. 6) The cytoskeletal protein ankyrin, which is known to bind to Na+-K+-ATPase, also stimulates enzyme activity, whereas BSA is without effect; the effects of adducin and ankyrin when acting together are not additive. 7) Pig kidney medulla microsomes appear to contain endogenous adducin; in contrast with purified pig kidney Na+-K+-ATPase, which does not contain adducin, added adducin stimulates the Na+-K+-ATPase activity of microsomes only about one-half as much as that of purified Na+-K+-ATPase. Our findings strongly imply the existence of a direct and specific interaction between adducin and Na+-K+-ATPase in vitro and also suggest the possibility of such an interaction in intact renal membranes.
α-Adrenergic vasoreactivity of canine intrapulmonary bronchial arteries in pacing-induced heart failure
Authors: McMillon Ronald K. , Townsley Mary I.
Published: December 2017

We hypothesized that pacing-induced congestive heart failure alters α-adrenergic constriction in intrapulmonary bronchial arteries. Cumulative dose responses to norepinephrine (NE), phenylephrine (PE), acetylcholine (ACh) and sodium nitroprusside (SNP) were determined in pressurized vessel segments. ED50values for NE and PE were higher for control (−5.34 ± 0.09 and −4.27 ± 0.08 M, respectively) vs. paced (−5.73 ± 0.10 and −5.06 ± 0.28 M, respectively) groups. Prazosin increased the ED50 values for NE and PE in both control and paced groups. Yohimbine decreased NE ED50 in the control group only. Endothelium removal or nitric oxide synthase (NOS) inhibition decreased control but not paced NE ED50. Maximum vasodilation and sensitivity (i.e., −ED50 values) were decreased for ACh but were similar for SNP in paced vs. control groups. Secondary segments were more reactive than paired primary segments in both groups, although pacing effects on ED50 were unrelated to branching order. In conclusion, adrenergic constriction of canine intrapulmonary bronchial arteries is predominantly mediated via α1-adrenoreceptors and is enhanced after pacing. Endothelium-derived relaxing factor(s) normally opposes α-adrenergic vasoconstriction but not after pacing in this vasculature.
Miconazole represses CO2-induced pial arteriolar dilation only under selected circumstances
Authors: Pelligrino Dale A. , Santizo Roberto A. , Wang Qiong
Published: December 2017

Previous experimental findings have led to the suggestion that guanosine 3′,5′-cyclic monophosphate (cGMP) plays a permissive role in hypercapnic cerebral vasodilation. However, we recently reported that the technique used to reveal a permissive role for cGMP [cGMP repletion in the presence of nitric oxide synthase (NOS) inhibition] created a situation where CO2reactivity was normalized but where different mechanisms (i.e., K+channels) participated in the response. In the present study, we examined whether that nascent K+-channel dependence is related in any way to an increase in the influence of the miconazole-inhibitable cytochrome P-450 epoxygenase pathway. Using intravital microscopy and a closed cranial window system in adult rats, we measured pial arteriolar diameters during normo- and hypercapnia, first in the absence and then in the presence of a neuronal NOS (nNOS) inhibitor [7-nitroindazole (7-NI)]. This was followed by suffusion of a cGMP analog and then cGMP plus miconazole. Separate groups of rats were used to evaluate whether miconazole either alone or in the presence of 8-bromoguanosine 3′,5′-cyclic monophosphate (8-BrcGMP) or its vehicle (0.1% ethanol) had any effect on CO2reactivity and whether miconazole affected K+-channel opener-induced dilations. Hypercapnic (arterial[Formula: see text], ≅65 mmHg) pial arteriolar dilations, as expected, were reduced by 70–80% with 7-NI and restored with cGMP repletion. CO2reactivity was again attenuated after miconazole introduction. Miconazole, with and without 8-BrcGMP, and its vehicle had no influence on pial arteriolar CO2reactivity in the absence of nNOS inhibition combined with cGMP repletion. Miconazole alone also did not affect vasodilatory responses to K+-channel openers. Thus present results suggest that the nascent K+-channel dependence of the hypercapnic response found in our earlier study may be related to increased epoxygenase activity. The specific reasons why the pial arteriolar CO2reactivity gains a K+-channel and epoxygenase dependence only under conditions of nNOS inhibition and cGMP restoration remain to be identified. These findings again call into question the interpretations applied to data collected in studies evaluating potential permissive actions of cGMP or NO.
Depolarization-mediated inhibition of Ca2+entry in endothelial cells
Authors: Wang Xiaodong , van Breemen Cornelis
Published: December 2017

The effect of extracellular Cl−in regulating ACh-induced Ca2+entry into freshly isolated rabbit aortic endothelial cells was studied using Ca2+-sensitive fluorescence microscopy and patch-clamp electrophysiology. After ACh caused transient Ca2+release in Ca2+-free medium, readdition of 3 mM Ca2+to the bath maintained Ca2+entry. Removal of extracellular Cl−abolished the plateau phase in Ca2+signal and inhibited divalent cation entry. However, in the presence of the K+ionophore valinomycin, removal of Cl−had no effect on the Ca2+plateau. Under current-clamp conditions, substitution of gluconate for Cl−induced membrane depolarization. Under voltage clamp, with CsCl in the pipette, ACh activated a slowly developing Cl−current, which was blocked by SITS and 5-nitro-2-(3-phenylpropylamino)benzoic acid. Varying the membrane potential by utilizing different extracellular K+concentrations in the presence of 5 μM valinomycin demonstrated that depolarization blocked ACh-stimulated Mn2+entry. These data suggest that ACh-induced Ca2+entry in freshly isolated endothelial cells requires the presence of extracellular Cl−to maintain a polarized membrane potential.
Emotional stress induces immediate-early gene expression in rat heart via activation of α- and β-adrenoceptors
Authors: Ueyama Takashi , Yoshida Ken-Ichi , Senba Emiko
Published: December 2017

We have studied the adrenergic mechanisms of immediate-early gene (IEG) induction in the discrete types of cardiac cells with the use of in situ hybridization histochemistry in an immobilization-stress model in conscious rats. Expression of c- fos, fos B, c- jun, jun B, NGFI-A, and NGFI-B mRNA was rapidly upregulated in the endothelial, myocardial, and smooth muscle cells of coronary vessels by 15–45 min after the onset of immobilization. Simultaneous blockade of both α- and β-adrenoceptors completely abolished expression of IEGs in these cardiac cells. Application of an α-agonist or β-agonist alone to the perfused rat heart under constant pressure elicited the upregulation of IEGs in a fashion similar to that of emotional stress. These data suggest that activation of either α- or β-adrenoceptor is sufficient to evoke expression of these genes and that there may be cross talk in signal transduction downstream from α- and β-adrenoceptors in cardiac cells.
Impaired wound healing and angiogenesis in eNOS-deficient mice
Authors: Lee Paul C. , Salyapongse A. Neil , Bragdon Gwynne A. , Shears Larry L. , Watkins Simon C. , Edington Howard D. J. , Billiar Timothy R.
Published: December 2017

A role for nitric oxide (NO) in wound healing has been proposed; however, the absolute requirement of NO for wound healing in vivo and the contribution of endothelial NO synthase (eNOS) have not been determined. Experiments were carried out using eNOS gene knockout (KO) mice to determine the requirement for eNOS on wound closure and wound strength. Excisional wound closure was significantly delayed in the eNOS KO mice (29.4 ± 2.2 days) compared with wild-type (WT) controls (20.2 ± 0.4 days). At 10 days, incisional wound tensile strength demonstrated a 38% reduction in the eNOS KO mice. Because effective wound repair requires growth factor-stimulated angiogenesis, in vitro and in vivo angiogenesis assays were performed in the mice to assess the effects of eNOS deficiency on angiogenesis. Endothelial cell sprouting assays confirmed in vitro that eNOS is required for proper endothelial cell migration, proliferation, and differentiation. Aortic segments harvested from eNOS KO mice cultured with Matrigel demonstrated a significant reduction in endothelial cell sprouting and [3H]thymidine incorporation compared with WT mice at 5 days. Capillary ingrowth into subcutaneously implanted Matrigel plugs was significantly reduced in eNOS KO mice (2.67 ± 0.33 vessels/plug) compared with WT mice (10.17 ± 0.79 vessels/plug). These results clearly show that eNOS plays a significant role in facilitating wound repair and growth factor-stimulated angiogenesis.
Transmural pressure induces matrix-degrading activity in porcine arteries ex vivo
Authors: Chesler Naomi C. , Ku David N. , Galis Zorina S.
Published: December 2017

Extracellular matrix components must be degraded and resynthesized for vascular remodeling to occur. We hypothesized that the hemodynamic environment regulates activity of matrix metalloproteinases (MMPs), the primary agents for in vivo matrix degradation, during vascular remodeling in response to changes in transmural pressure and shear stress. Pathological hemodynamic conditions were reproduced in an ex vivo system in which we maintained porcine carotid arteries for 24 and 48 h. Total levels of MMP-2 and MMP-9 extracted from tissue homogenates and analyzed by SDS-PAGE zymography were stimulated by transmural pressure and were unaffected by shear stress changes. Degradation of two specific gelatinase substrates, gelatin and elastin, increased with increasing pressure, but the degradation was not affected by shear stress changes in tissue specimens analyzed using in situ zymography (gelatin) and fluorescent measurement of endogenous elastin degradation (elastin). Our results suggest that transmural pressure activates at least two members of the MMP family and that activity of these enzymes is accompanied by degradation of matrix components, effects that may be implicated in hypertensive vascular remodeling.
Impaired lusitropy-frequency in the aging mouse: role of Ca2+-handling proteins and effects of isoproterenol
Authors: Lim Chee Chew , Liao Ronglih , Varma Niraj , Apstein Carl S.
Published: December 2017

We examined the relationship between age-associated lusitropic impairment, heart rate, and Ca2+-handling proteins and assessed the efficacy of increasing left ventricular (LV) relaxation via β-adrenergic stimulation in adult and aging mouse hearts. LV function was measured in isolated, isovolumic blood-perfused hearts from adult (5 mo), old (24 mo), and senescent (34 mo) mice. Hearts were paced from 5 to 10 Hz, returned to 7 Hz, exposed to 10−6 M isoproterenol, and paced again from 7 to 10 Hz. Age-related alterations in Na+/Ca2+exchanger (NCX), sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a), and phospholamban (PLB) levels were assessed by immunoblot. Despite preserved contractile performance, aging caused impaired lusitropy. Increased pacing caused an elevation in end-diastolic pressure that progressively worsened with age. The time constant of isovolumic pressure decay (τ) was significantly prolonged in old and senescent hearts compared with adults. Relative to adult hearts, the SERCA2a-to-PLB ratios were reduced 68 and 69%, and NCX were reduced 37 and 58% in old and senescent hearts, respectively. Isoproterenol completely reversed the age-associated lusitropic impairments. These data suggest that impaired lusitropy in aging mouse hearts is related to a decreased rate of cytosolic Ca2+ removal and that accelerating SR Ca2+ resequestration via β-adrenergic stimulation can reverse this impairment.
Effects of methionine on endogenous antioxidants in the heart
Authors: Seneviratne Charita K. , Li Timao , Khaper Neelam , Singal Pawan K.
Published: December 2017

The deficiency of methionine, an essential amino acid, is associated with cardiovascular lesions. Because different types of cardiac pathologies are caused by a decrease in antioxidants, we examined the effects of methionine on myocardial antioxidant enzymes in hemodynamically assessed rats that were treated with methionine (10 mg/ml) in drinking water for 12, 24, and 48 h. Glutathione peroxidase (GSHPx) activity was significantly increased to 150.5 ± 12.2 and 191.7 ± 13.7% of the control value at 12 and 24 h, respectively, followed by a decline to 120 ± 24.6% at 48 h. The mRNA levels of GSHPx at these time points were 151.2 ± 12.0, 218.7 ± 35.3, and 173.5 ± 25.2%, respectively. Superoxide dismutase (SOD) activity was 144.3 ± 3.7, 114.3 ± 10.1, and 143.1 ± 11.2% at 12, 24, and 48 h, respectively. Catalase (Cat) activity was 272.4 ± 5.4, 237.8 ± 16.6, and 224.1 ± 17.3% of the control value. The expression of Cat and SOD mRNA was unchanged at 12, 24, and 48 h. The lipid peroxidation was decreased by 24.4 ± 11.2, 54.9 ± 0.1, and 6.4 ± 2.1% at 12, 24, and 48 h, respectively. Methionine had no effect on the ventricular or aortic pressures, heart rate, and myocardial glutathione levels at any of the time points. The study shows that methionine has a significant effect on the myocardial antioxidant enzyme activities, and only changes in GSHPx enzyme activity correlated with the mRNA changes. These antioxidant changes may have a role in the beneficial effects of methionine in pathological rather than physiological conditions.
Dynamic counterbalance between direct and indirect vagal controls of atrioventricular conduction in cats
Authors: Chen Shi-Liang , Kawada Toru , Inagaki Masashi , Shishido Toshiaki , Miyano Hiroshi , Sato Takayuki , Sugimachi Masaru , Takaki Hiroshi , Sunagawa Kenji
Published: December 2017

The vagal system regulates the atrioventricular conduction time ( T AV) via two opposing mechanisms: a direct effect on the atrioventricular node and an indirect effect through changes in heart period ( T AA). To evaluate how dynamic vagal activation affects T AV, we stimulated the vagal nerve with frequency-modulated Gaussian white noise and estimated the transfer function from vagal stimulation to the T AV response under conditions of no pacing and constant pacing in anesthetized cats. The effect of changes in T AA on T AV was estimated by a random-pacing protocol. The transfer function from vagal stimulation to T AV has low-pass filter characteristics. Constant pacing increased the maximum step response in T AV(2.4 ± 1.2 vs. 6.3 ± 2.2 ms/Hz, P < 0.01). The time constant did not differ between the vagal effect on T AV and that on T AA (2.9 ± 1.2 vs. 2.3 ± 0.5 s). Because changes in T AA reciprocally affected T AVwithout significant delay, the direct and indirect effects were dynamically counterbalanced and exerted stable T AV transient response during vagal stimulation under normal sinus rhythm.
Modulation of myocardial function and [Ca2+] sensitivity by moderate hypothermia in guinea pig isolated hearts
Authors: Stowe David F. , Fujita Satoshi , An Jianzhong , Paulsen Richard A. , Varadarajan Srinivasan G. , Smart Steven C.
Published: December 2017

Cardiac hypothermia alters contractility and intracellular Ca2+concentration ([Ca2+]i) homeostasis. We examined how left ventricular pressure (LVP) is altered as a function of cytosolic [Ca2+]iover a range of extracellular CaCl2concentration ([CaCl2]e) during perfusion of isolated, paced guinea pig hearts at 37°C, 27°C, and 17°C. Transmural LV phasic [Ca2+] was measured using the Ca2+indicator indo 1 and calibrated (in nM) after correction was made for autofluorescence, temperature, and noncytosolic Ca2+. Noncytosolic [Ca2+]i, cytosolic diastolic and systolic [Ca2+]i, phasic [Ca2+]i, and systolic Ca2+released per beat (area Ca2+) were plotted as a function of 0.3–4.5 mM [CaCl2]e, and indexes of contractility [LVP, maximal rates of LVP development (+dLVP/d t) and relaxation (−dLVP/d t), and the integral of the LVP curve per beat (LVParea)] were plotted as a function of [Ca2+]i. Hypothermia increased systolic [Ca2+]iand slightly changed systolic LVP but increased diastolic LVP and [Ca2+]i. The relationship of diastolic and noncytosolic [Ca2+] to [CaCl2]ewas shifted upward at 17°C and 27°C, whereas that of phasic [Ca2+]ito [CaCl2]ewas shifted upward at 17°C but not at 27°C. The relationships of phasic [Ca2+]ito developed LVP, +dLVP/d t, and LVPareawere progressively reduced by hypothermia so that maximal Ca2+-activated LVP decreased and hearts were desensitized to Ca2+. Thus mild hypothermia modestly increases diastolic and noncytosolic Ca2+with little effect on systolic Ca2+or released (area) Ca2+, whereas moderate hypothermia markedly increases diastolic, noncytosolic, peak systolic, and released Ca2+and results in reduced maximal Ca2+-activated LVP and myocardial sensitivity to systolic Ca2+.
Intramyocardial vascular volume distribution studied by synchrotron radiation-excited X-ray fluorescence
Authors: Nakajima Yukiko , Akizuki Noboru , Kimura Yoko , Kohguchi Hiroki , Tanaka Akira , Chujo Mitsuaki , Hattan Naoichiro , Shinozaki Yoshiro , Iida Atsuo , Handa Shunnosuke , Nakazawa Hiroe , Mori Hidezo
Published: December 2017

We evaluated the vascular volume distribution with fine resolution (0.1–1.3 mg myocardial tissue) in the sagittal plane of the left ventricle by using the microsphere filling method in 21 dogs. The coronary arterial volume density in the sagittal plane did not exhibit normal distribution and was characterized by variability among the outer-to-inner layers and within the layers (+2SD/−2SD > 80 times), and the median values in the layers ranged from 4.7 to 22.9 nl/mg myocardial tissue. The fractal analysis of vascular volume revealed a self-similar nature with a fractal dimension ( D value) similar to that of flow distribution (1.20 ± 0.05 and 1.24 ± 0.09 for vascular volume and flow distribution, respectively) and had a more marked variability than the flow. The correlation of the regional vascular volume between adjacent regions decreased as the distance increased. However, the correlation coefficients in the endocardial-to-epicardial direction were significantly higher than those in the anterior-to-posterior direction ( P < 0.05 by paired t-test). In conclusion, we determined intramyocardial vascular volume density in the sagittal plane, and the distribution revealed considerable variability, self-similarity, and asymmetry in the correlation among the adjacent regions. These observations could be related to the characteristics of the intramural coronary vascular network.
Toluidine blue O and methylene blue as endothelial redox probes in the intact lung
Authors: Audi Said H. , Olson Lars E. , Bongard Robert D. , Roerig David L. , Schulte Marie L. , Dawson Christopher A.
Published: December 2017

There is increasing evidence that the redox activities of the pulmonary endothelial surface may have important implications for the function of both lungs and blood. Because of the inherent complexity of intact organs, it can be difficult to study these activities in situ. Given the availability of appropriate indicator probes, the multiple-indicator dilution (MID) method is one approach for dealing with some aspects of this complexity. Therefore, the objectives of the present study were to 1) evaluate the potential utility of two thiazine redox indicators, methylene blue (MB) and toluidine blue O (TBO), as MID electron acceptor probes for in situ pulmonary endothelium and 2) develop a mathematical model of the pulmonary disposition of these indicators as a tool for quantifying their reduction on passage through the lungs. Experiments were carried out using isolated rabbit lungs perfused with physiological salt solution with or without plasma albumin over a range of flow rates. A large fraction of the injected TBO disappeared from the perfusate on passage through the lungs. The reduction of its oxidized, strongly polar, relatively hydrophilic blue form to its colorless, highly lipophilic reduced form was revealed by the presence of the reduced form in the venous effluent when plasma albumin was included in the perfusate. MB was also lost from the perfusate, but the fraction was considerably smaller than for TBO. A distributed-in-space-and-time model was developed to estimate the reduction rate parameter, which was ∼29 and 1.0 ml/s for TBO and MB, respectively, and almost flow rate independent for both indicators. The results suggest the utility particularly of TBO as an electron acceptor probe for MID studies of in situ pulmonary endothelium and of the model for quantitative evaluation of the data.
The 400 microsphere per piece “rule” does not apply to all blood flow studies
Authors: Polissar Nayak L. , Stanford Derek C. , Glenny Robb W.
Published: December 2017

Microsphere experiments are useful in measuring regional organ perfusion as well as heterogeneity of blood flow within organs and correlation of perfusion between organ pieces at different time points. A 400 microspheres/piece “rule” is often used in planning experiments or to determine whether experiments are valid. This rule is based on the statement that 400 microspheres must lodge in a region for 95% confidence that the observed flow in the region is within 10% of the true flow. The 400 microspheres precision rule, however, only applies to measurements of perfusion to a single region or organ piece. Examples, simulations, and an animal experiment were carried out to show that good precision for measurements of heterogeneity and correlation can be obtained from many experiments with <400 microspheres/piece. Furthermore, methods were developed and tested for correcting the observed heterogeneity and correlation to remove the Poisson “noise” due to discrete microsphere measurements. The animal experiment shows adjusted values of heterogeneity and correlation that are in close agreement for measurements made with many or few microspheres/piece. Simulations demonstrate that the adjusted values are accurate for a variety of experiments with far fewer than 400 microspheres/piece. Thus the 400 microspheres rule does not apply to many experiments. A “rule of thumb” is that experiments with a total of at least 15,000 microspheres, for all pieces combined, are very likely to yield accurate estimates of heterogeneity. Experiments with a total of at least 25,000 microspheres are very likely to yield accurate estimates of correlation coefficients.