bluuebunny/crossref_metadata_2025 huggingface dataset:
P2Y1, P2Y2, P2Y4, and P2Y6receptors are coupled to Rho and Rho kinase activation in vascular myocytes
Authors: Sauzeau Vincent , le Jeune Hélène , Cario-Toumaniantz Chrystelle , Vaillant Nathalie , Gadeau Alain-Pierre , Desgranges Claude , Scalbert Elizabeth , Chardin Pierre , Pacaud Pierre , Loirand Gervaise
Published: December 2017

In the cardiovascular system, activation of ionotropic (P2X receptors) and metabotropic (P2Y receptors) P2 nucleotide receptors exerts potent and various responses including vasodilation, vasoconstriction, and vascular smooth muscle cell proliferation. Here we examined the involvement of the small GTPase RhoA in P2Y receptor-mediated effects in vascular myocytes. Stimulation of cultured aortic myocytes with P2Y receptor agonists induced an increase in the amount of membrane-bound RhoA and stimulated actin cytoskeleton organization. P2Y receptor agonist-induced actin stress fiber formation was inhibited by C3 exoenzyme and the Rho kinase inhibitor Y-27632. Stimulation of actin cytoskeleton organization by extracellular nucleotides was also abolished in aortic myocytes expressing a dominant negative form of RhoA. Extracellular nucleotides induced contraction and Y-27632-sensitive Ca2+sensitization in aortic rings. Transfection of Swiss 3T3 cells with P2Y receptors showed that Rho kinase-dependent actin stress fiber organization was induced in cells expressing P2Y1, P2Y2, P2Y4, or P2Y6receptor subtypes. Our data demonstrate that P2Y1, P2Y2, P2Y4, and P2Y6receptor subtypes are coupled to activation of RhoA and subsequently to Rho-dependent signaling pathways.
The Rho effector, PKN, regulates ANF gene transcription in cardiomyocytes through a serum response element
Authors: Morissette Michael R. , Sah Valerie P. , Glembotski Christopher C. , Brown Joan Heller
Published: December 2017

The low-molecular-weight GTP-binding protein RhoA mediates hypertrophic growth and atrial natriuretic factor (ANF) gene expression in neonatal rat ventricular myocytes. Neither the effector nor the promoter elements through which Rho exerts its regulatory effects on ANF gene expression have been elucidated. When constitutively activated forms of Rho kinase and two protein kinase C-related kinases, PKN (PRK1) and PRK2, were compared, only PKN generated a robust stimulation of a luciferase reporter gene driven by a 638-bp fragment on the ANF promoter. This ANF promoter fragment contains a proximal serum response element (SRE) and an Sp-1-like element required for the transcriptional response to phenylephrine (PE). This response was inhibited by dominant negative Rho. The ability of dominant negative Rho to inhibit the response to PE and the ability of PKN to stimulate ANF reporter gene expression were both lost when the SRE was mutated. Mutation of the Sp-1-like element also attenuated the response to PKN. A minimal promoter driven by ANF SRE sequences was sufficient to confer Rho- and PKN-mediated gene expression. Interestingly, PKN preferentially stimulated the ANF versus the c- fos SRE reporter gene. Thus PKN and Rho are able to regulate transcriptional activation of the ANF SRE by a common element that could implicate PKN as a downstream effector of Rho in transcriptional responses associated with hypertrophy.
Gi-dependent suppression of β1-adrenoceptor effects in ventricular myocytes from NE-treated guinea pigs
Authors: Ranu Hardeep K. , Mak Judith C. W. , Barnes Peter J. , Harding Sian E.
Published: December 2017

It has been suggested that there is a preferential coupling in heart muscle between the inhibitory G protein (Gi) and the β2-subtype of the β-adrenergic receptor (β-AR), since pertussis toxin (which inactivates Gi) reveals latent β2-ARs in rat and mouse myocytes. We have previously shown that guinea pigs treated with norepinephrine (NE) for 7 days have myocytes that are desensitized to β-AR-agonist stimulation, and that pertussis toxin restores these responses. The purpose of the present investigation was to determine whether pertussis toxin specifically upregulated β2-ARs in myocytes from NE-treated guinea pigs. The sole β-AR subtype in control guinea pig myocytes was confirmed as β1-AR by radioligand binding, single-cell autoradiography, and concentration-response curves to isoproterenol in contracting myocytes. In contrast, a minor pool of β2-ARs was observed in rat myocytes by use of the same methods. NE treatment decreased the maximum isoproterenol response (relative to high Ca2+) from 0.89 ± 0.06 to 0.58 ± 0.08 ( n = 7, P < 0.01) and the pD2(−log EC50) from 8.8 ± 0.2 to 7.5 ± 0.2 ( n = 7, P < 0.01). Pertussis toxin treatment increased the isoproterenol-to-Ca2+ratio to 0.88 ± 0.04 ( n = 6, P < 0.05) and the pD2to 8.6 ± 0.3 ( P < 0.01). This was not mediated by increases in either number or function of β2-ARs. Giis therefore able to modulate β1-AR responses in guinea pig myocytes.
Structural and functional remodeling of skeletal muscle microvasculature is induced by simulated microgravity
Authors: Delp Michael D. , Colleran Patrick N. , Wilkerson M. Keith , McCurdy Matthew R. , Muller-Delp Judy
Published: December 2017

Hindlimb unloading of rats results in a diminished ability of skeletal muscle arterioles to constrict in vitro and elevate vascular resistance in vivo. The purpose of the present study was to determine whether alterations in the mechanical environment (i.e., reduced fluid pressure and blood flow) of the vasculature in hindlimb skeletal muscles from 2-wk hindlimb-unloaded (HU) rats induces a structural remodeling of arterial microvessels that may account for these observations. Transverse cross sections were used to determine media cross-sectional area (CSA), wall thickness, outer perimeter, number of media nuclei, and vessel luminal diameter of feed arteries and first-order (1A) arterioles from soleus and the superficial portion of gastrocnemius muscles. Endothelium-dependent dilation (ACh) was also determined. Media CSA of resistance arteries was diminished by hindlimb unloading as a result of decreased media thickness (gastrocnemius muscle) or reduced vessel diameter (soleus muscle). ACh-induced dilation was diminished by 2 wk of hindlimb unloading in soleus 1A arterioles, but not in gastrocnemius 1A arterioles. These results indicate that structural remodeling and functional adaptations of the arterial microvasculature occur in skeletal muscles of the HU rat; the data suggest that these alterations may be induced by reductions in transmural pressure (gastrocnemius muscle) and wall shear stress (soleus muscle).
Remote arteriolar dilations in response to muscle contraction under capillaries
Authors: Cohen Kenneth D. , Berg Bradley R. , Sarelius Ingrid H.
Published: December 2017

In hamster cremaster muscle, it has been shown previously that contraction of skeletal muscle fibers underlying small groups of capillaries (modules) induces dilations that are proportional to metabolic rate in the two arteriolar generations upstream of the stimulated capillaries (Berg BR, Cohen KD, and Sarelius IH. Am J Physiol Heart Circ Physiol 272: H2693–H2700, 1997). These remote dilations were hypothesized to be transmitted via gap junctions and not perivascular nerves. In the present study, halothane (0.07%) blocked dilation in the module inflow arteriole, and dilation in the second arteriolar generation upstream, the branch arteriole, was blocked by both 600 mosM sucrose and halothane but not tetrodotoxin (2 μM). Dilations in both arterioles were not blocked by the gap junction uncoupler 18-β-glycyrrhetinic acid (40 μM), and 80 mM KCl did not block dilation of the module inflow arteriole. These data implicate a gap junctional-mediated pathway insensitive to 18-β-glycyrrhetinic acid in dilating the two arterioles upstream of the capillary module during “remote” muscle contraction. Dilation in the branch arteriole, but not the module inflow arteriole, was attenuated by 100 μM N ω-nitro-l-arginine. Thus selective contraction of muscle fibers underneath capillaries results in dilations in the upstream arterioles that have characteristics consistent with a signal that is transmitted along the vessel wall through gap junctions, i.e., a conducted vasodilation. The observed insensitivities to 18-β-glycyrrhetinic acid, to KCl, and to N ω-nitro-l-arginine suggest, however, that there are multiple signaling pathways by which remote dilations can be initiated in these microvessels.
Identification of ecto-PKC on surface of human platelets: role in maintenance of latent fibrinogen receptors
Authors: Babinska Anna , Hogan Michael V. , Sobocki Tomasz , Sobocka Malgorzata B. , Ehrlich Yigal H. , Kornecki Elizabeth
Published: December 2017

Human platelets express a protein phosphorylation system on their surface. A specific protein kinase C (PKC) antibody, monoclonal antibody (MAb) 1.9, which binds to the catalytic domain of PKC and inhibits its activity, causes the aggregation of intact platelets while inhibiting the phosphorylation of platelet surface proteins. Photoaffinity labeling with 100 nM 8-azido-[α32P]ATP identified this ecto-PKC as a single surface protein of 43 kDa sensitive to proteolysis by extracellular 0.0005% trypsin. Inhibition of the binding of 8-azido-[α32P]ATP to the 43-kDa surface protein by MAb 1.9 identified this site as the active domain of ecto-PKC. Covalent binding of the azido-ATP molecule to the 43-kDa surface protein inhibited the phosphorylative activity of the platelet ecto-PKC. Furthermore, PKC pseudosubstrate inhibitory peptides directly induced the aggregation of platelets and inhibited azido-ATP binding to the 43-kDa protein. Platelet aggregation induced by MAb 1.9 and by PKC inhibitory peptides required the presence of fibrinogen and resulted in an increase in the level of intracellular free calcium concentration. This increase in intracellular free calcium concentration induced by MAb 1.9 was found to be dependent on the binding of fibrinogen to activated GPIIb/IIIa integrins, suggesting that MAb 1.9 causes Ca2+flux through the fibrinogen receptor complex. We conclude that a decrease in the state of phosphorylation of platelet surface proteins caused by inhibition of ecto-PKC results in membrane rearrangements that can induce the activation of latent fibrinogen receptors, leading to platelet aggregation. Accordingly, the maintenance of a physiological steady state of phosphorylation of proteins on the platelet surface by ecto-PKC activity appears to be one of the homeostatic mechanisms that maintain fibrinogen receptors of circulating platelets in a latent state that cannot bind fibrinogen.
Endothelial dysfunction does not require loss of endothelial nitric oxide synthase
Authors: Giraldez Roberto R. , Panda Alexander , Zweier Jay L.
Published: December 2017

Whereas altered nitric oxide (NO ⋅) formation from endothelial nitric oxide synthase (NOS) causes impaired vascular reactivity in a number of cardiovascular diseases, questions remain regarding how endothelial injury results in impaired NO ⋅ formation. It is unknown if loss of NOS expression or activity is required or if other factors are involved. Detergent treatment has been used to induce endothelial dysfunction. Therefore, NOS and NO ⋅ synthesis were characterized in a rat heart model of endothelial injury and dysfunction induced by the detergent Triton X-100. Cardiac NO ⋅ formation was directly measured by electron paramagnetic resonance spectroscopy. NOS activity was determined by thel-[14C]arginine conversion assay. Western blots and immunohistology were applied to define the amounts of NOS present in heart tissue before and after Triton treatment. Immunoelectron microscopy was performed to assess intracellular NOS distribution. A short bolus of Triton X-100, 0.25%, abolished responses to histamine and calcium ionophore while preserving response to nitroprusside. Complete blockade of NO ⋅ generation occurred after Triton treatment, but NOS activity assayed with addition of exogenous substrate and cofactors was unchanged, and identical 135-kDa NOS bands were seen on Western blots, indicating that NOS was not removed from the heart or structurally damaged by Triton. Immunohistochemistry showed no change in NOS localization after Triton treatment, and immunoelectron microscopy revealed similar NOS distribution in the plasma membrane and intracellular membranes. These results demonstrate that the endothelial dysfunction was due to decreased NO ⋅ synthesis but was not caused by loss or denaturation of NOS. Thus endothelial dysfunction due to mild endothelial membrane injury may occur in the presence of active NOS and is triggered by loss of NOS substrates or cofactors.
A self-powered constant infusion device for use in unrestrained rats
Authors: Brand Paul H. , Qi Nianning , Metting Patricia J. , Britton Steven L.
Published: December 2017

We developed a device that delivers fluid through a catheter at a constant rate and can be used in conscious animals to solve a variety of problems. For example, this device can be used for delivering drugs and maintaining intravascular catheter patency. The device provides infusions at low flows (1.0–1.5 ml/day), so that experimental agents may be administered with minimal volume loading of the rat. Arterial and venous catheter patency is maintained by infusion of heparinized saline through indwelling catheters attached to the device. The catheters exit from the rat in the intrascapular area and are routed through a protective spring to the device, which is suspended above the cage. The catheters may be attached to pressure transducers, blood may be sampled, and injections or infusions may be made without disturbing the rat. Because the device is self-contained, it can be suspended by a fluid-free swivel that rotates through 360°, providing minimal restraint. The device has been used successfully to measure arterial and central venous blood pressures in two studies using rats.
Modulation of arrhythmias by isoproterenol in a rabbit heart model of d-sotalol-induced long Q-T intervals
Authors: Spear Joseph F. , Moore E. Neil
Published: December 2017

Sympathetic influences have been implicated in arrhythmias associated with both congenital and acquired long Q-T intervals. We recorded epicardial electrograms, a left ventricular endocardial monophasic action potential (MAP), and a bipolar electrocardiogram in 23 isolated rabbit hearts. Spontaneous focal arrhythmias appeared within 8–18 min following 92 μM d-sotalol in 15 of 23 hearts. The epicardial activation-recovery interval was shorter at baseline and increased to a significantly greater degree after d-sotalol administration in the hearts that developed focal activity. The standard deviation of the activation-recovery interval of the epicardial sites also increased. With the addition of 0.01 μM isoproterenol, the incidence of focal activity increased, and its mean cycle length was shortened by 7%. Also, myocardial recovery time in the epicardium was shortened to a greater degree than the endocardial MAP duration. It did not alter local epicardial heterogeneity of recovery but did increase the regional dispersion between epicardial recovery times, and the endocardial MAP duration. Therefore, β-adrenergic stimulation in the presence of d-sotalol favors the appearance of arrhythmias by increasing the propensity for closely coupled focal activity and the temporal dispersion of recovery.
Biochemical mechanism(s) of stunning in conscious dogs
Authors: Lüss Hartmut , Meissner Andreas , Rolf Norbert , Van Aken Hugo , Bokník Peter , Kirchhefer Uwe , Knapp Jörg , Läer Stephanie , Linck Bettina , Lüss Iva , Müller Frank U. , Neumann Joachim , Schmitz Wilhelm
Published: December 2017

The mechanism(s) underlying contractile dysfunction in cardiac stunning is not completely understood. The expression and/or the phosphorylation state of cardiac Ca2+homoeostasis-regulating proteins might be altered in stunning. We tested this hypothesis in a well-characterized model of stunning. Conscious dogs were chronically instrumented, and the left anterior descending artery (LAD) was occluded for 10 min. Thereafter, reperfusion of the LAD was initiated. Tissues from reperfused LAD (stunned) and Ramus circumflexus (control) areas were obtained when left ventricular regional wall thickening fraction had recovered by 50%. Northern and Western blotting revealed no differences in the expression of the following genes: phospholamban, calsequestrin, sarco(endo)plasmic reticulum Ca2+-ATPase 2a, and the inhibitory subunit of troponin I (TnI). However, the phosphorylation state of TnI and phospholamban were reduced in the LAD area. Fittingly, cAMP levels were reduced by 28% ( P < 0.05). It is concluded that the contractile dysfunction in cardiac stunning might be mediated in part by decreased levels of cAMP and subsequently a reduced phosphorylation state of phospholamban and TnI.
Free radicals upregulate complement expression in rabbit isolated heart
Authors: Tanhehco Elaine J. , Yasojima Koji , McGeer Patrick L. , Washington Ruth A. , Lucchesi Benedict R.
Published: December 2017

Both free radicals and complement activation can injure tissue. Our study determined whether free radicals alter complement production by the myocardium. Isolated hearts from New Zealand White rabbits were perfused on a Langendorff apparatus and exposed to xanthine (X; 100 μM) plus xanthine oxidase (XO; 8 mU/ml) (X/XO). The free radical-generating system significantly ( P < 0.05) increased C1q and also increased C1r, C3, C8, and C9 transcription compared with controls. Immunohistological examination revealed augmented membrane attack complex deposition on X/XO-treated tissue. X/XO-treated hearts also exhibited significant ( P < 0.05) increases in coronary perfusion pressure and left ventricular end-diastolic pressure and a decrease in left-ventricular developed pressure. N-(2-mercaptopropionyl)-glycine (3 mM), in conjunction with the superoxide dismutase mimetic SC-52608 (100 μM), significantly ( P < 0.05) reduced the upregulation of C1q, C1r, C3, C8, and C9 mRNA expression elicited by X/XO. The antioxidants also ameliorated the deterioration in function caused by X/XO. Local complement activation may represent a mechanism by which free radicals mediate tissue injury.
Effects of exercise training on LV performance and mortality in a murine model of dilated cardiomyopathy
Authors: Spencer Kirk T. , Collins Keith , Korcarz Claudia , Fentzke Richard , Lang Roberto M. , Leiden Jeffrey M.
Published: December 2017

Dilated cardiomyopathy (DC) is a leading cause of cardiovascular morbidity, and nonpharmacological therapies, such as exercise training, have been suggested. The effects of exercise on left ventricular (LV) function and mortality remain controversial. Using a recently described murine model of DC, which involves a dominant-negative form of the cAMP response element binding protein (CREB) transcription factor (CREBA133) under the control of the cardiac myocyte-specific α-myosin heavy chain promoter, we sought to assess the effects of moderate-intensity exercise training on LV performance and mortality. Thirty-two transgenic mice were subjected to exercise training and compared with sedentary controls. There was progressive enlargement in LV dimensions in both the sedentary and exercise-trained mice. LV performance was progressively impaired, and exercise training did not prevent this decline. The sedentary CREBA133 mice displayed a significantly increased rate of death, and exercise training did not prevent or delay this excess mortality. The CREBA133 murine model of inherited DC demonstrated progressive ventricular dilatation and dysfunction with increased mortality, which was not altered with 12 wk of moderate-intensity exercise training.
Microvascular dilation in response to occlusion: a coordinating role for conducted vasomotor responses
Authors: Dora Kim A. , Damon David N. , Duling Brian R.
Published: December 2017

In rat cremasteric microcirculation, mechanical occlusion of one branch of an arteriolar bifurcation causes an increase in flow and vasodilation of the unoccluded daughter branch. This dilation has been attributed to the operation of a shear stress-dependent mechanism in the microcirculation. Instead of or in addition to this, we hypothesized that the dilation observed during occlusion is the result of a conducted signal originating distal to the occlusion. To test this hypothesis, we blocked the ascending spread of conducted vasomotor responses by damaging the smooth muscle and endothelial cells in a 200-μm segment of second- or third-order arterioles. We found that a conduction blockade eliminated or diminished the occlusion-associated increase in flow through the unoccluded branch and abolished or strongly attenuated the vasodilatory response in both vessels at the branch. We also noted that vasodilations induced by ACh (10−4 M, 0.6 s) spread to, but not beyond, the area of damage. Taken together, these data provide strong evidence that conducted vasomotor responses have an important role in coordinating blood flow in response to an arteriolar occlusion.
P2X4 receptors mediate ATP-induced calcium influx in human vascular endothelial cells
Authors: Yamamoto Kimiko , Korenaga Risa , Kamiya Akira , Qi Zhi , Sokabe Masahiro , Ando Joji
Published: December 2017

ATP induces Ca2+ influx across the cell membrane and activates release from intracellular Ca2+ pools in vascular endothelial cells (ECs). Ca2+ signaling leads to the modification of a variety of EC functions, including the production of vasoactive substances such as nitric oxide and prostacyclin. However, the molecular mechanisms for ATP-induced Ca2+ influx in ECs have not been thoroughly clarified. Here we demonstrate evidence that a P2X4receptor for an ATP-gated cation channel is predominantly expressed in human ECs and is involved in the ATP-induced Ca2+ influx. Northern blot analysis distinctly showed the expression of P2X4 mRNA in human ECs cultured from the umbilical vein, aorta, pulmonary artery, and skin microvessels. Competitive PCR revealed that P2X4 mRNA expression was much higher in ECs than was the expression of other subtypes, including P2X1, P2X3, P2X5, and P2X7. Treatment of ECs with antisense oligonucleotides designed to target the P2X4 receptor decreased the P2X4 mRNA and protein levels to ∼25% of control levels and markedly prevented the ATP-induced Ca2+ influx.
Calcium-activated potassium channels and nitric oxide coregulate estrogen-induced vasodilation
Authors: Rosenfeld Charles R. , White Richard E. , Roy Tim , Cox Blair E.
Published: December 2017

Nitric oxide synthase (NOS) contributes to estradiol-17β (E2β)-induced uterine vasodilation, but additional mechanisms are involved, and the cellular pathways remain unclear. We determined if 1) uterine artery myocytes express potassium channels, 2) E2β activates these channels, and 3) channel blockade plus NOS inhibition alters E2β-induced uterine vasodilation. Studies of cell-attached patches identified a 107 ± 7 pS calcium-dependent potassium channel (BKCa) in uterine artery myocytes that rapidly increased single-channel open probability 70-fold ( P < 0.05) after exposure to 100 nM E2β through an apparent cGMP-dependent mechanism. In ovariectomized nonpregnant ewes ( n = 11) with uterine artery flow probes and catheters, local BKCa blockade with tetraethylammonium (TEA; 0.05–0.6 mM) dose dependently inhibited E2β-induced uterine vasodilation ( n = 37, R = 0.77, P < 0.0001), with maximum inhibition averaging 67 ± 11%. Mean arterial pressure (MAP) and E2β-induced increases ( P ≤ 0.001) in heart rate (13%) and contralateral uterine blood flow (UBF, ∼5-fold) were unaffected. Local NOS inhibition plus BKCa blockade, using submaximal doses of nitro-l-arginine methyl ester (5 mg/ml) and TEA (0.3 mM), did not alter basal UBF but completely inhibited ipsilateral E2β-induced uterine vasodilation without affecting MAP and E2β-induced increases in contralateral UBF and heart rate. Acute E2β-mediated uterine vasodilation involves rapid activation of uterine artery BKCa and NOS, and the pathway for their interaction appears to include activation of guanylyl cyclase.
Effects of losartan treatment on cardiac autonomic control during volume loading in patients with DCM
Authors: Petretta M. , Spinelli L. , Marciano F. , Apicella C. , Vicario M. L. E. , Testa G. , Volpe M. , Bonaduce D.
Published: December 2017

This study evaluated the effect of angiotensin II receptor blockade on cardiac autonomic control adaptation and urine output in response to acute isotonic volume load in patients with idiopathic dilated cardiomyopathy (DCM) and asymptomatic to mildly symptomatic heart failure. Left ventricular volumes and heart rate variability measurements were assessed at baseline and during intravenous saline load in 14 patients before and after 2 mo of losartan treatment. After losartan treatment, blood pressure values were lower, whereas left ventricular ejection fraction was higher ( F = 79, P < 0.001), than before treatment. During saline load, ejection fraction decreased before losartan treatment ( F = 5.6, P< 0.05) but did not change after treatment. Urinary volume, unchanged during saline load in untreated patients, increased after losartan ( F = 9.38, P < 0.001). Time-domain measurements that represent vagal modulation of heart rate (root-mean-square successive differences and percentage of differences between successive R-R intervals >50 ms) decreased during saline load in untreated patients ( F = 3.1, P < 0.05 and F = 6.5, P < 0.01, respectively), but not after losartan. Similarly, a decrease in very low frequency ( F = 3.2, P < 0.05), low-frequency ( F = 2.9, P < 0.05), and high-frequency power ( F = 6.1, P < 0.01) after saline load was observed only in untreated patients. In patients with DCM, losartan treatment improves the cardiac autonomic adaptation and increases urine output in response to volume overload.
A comparison of transient outward currents in canine cardiac Purkinje cells and ventricular myocytes
Authors: Han Wei , Wang Zhiguo , Nattel Stanley
Published: December 2017

Although abnormalities in Purkinje cell (PC) repolarization are important causes of cardiac arrhythmias, the detailed properties of repolarizing currents in PCs are incompletely understood. We compared transient outward K+ current ( I to) in single PCs from canine false tendons with midmyocardial ventricular myocytes (VMs). I to reactivation was biexponential, with a similar rapid-phase time constant (30 ± 5 and 35 ± 4 ms for VM and PC, respectively) but a large, slow component in PCs with a much greater time constant than VM (1,427 ± 70 vs. 181 ± 24 ms, P < 0.001). Tetraethylammonium had no effect on VM I to but reversibly inhibited PC I to (IC50 = 2.4 ± 0.4 mM). PC I to was also more sensitive to 4-aminopyridine (IC50 = 50 ± 7 vs. 526 ± 49 μM in VM, P < 0.0001). H2O2slowed I to inactivation in PCs but did not affect VM I to. We conclude that PC I to shows significant differences from VM I to, with some features, such as tetraethylammonium sensitivity, that have been reported in neither cardiac I to of atrial or ventricular myocytes nor cloned K+ channel subunits (Kv1.4, Kv4.2, or Kv4.3) known to participate in cardiac I to.
Niacin protects the isolated heart from ischemia-reperfusion injury
Authors: Trueblood Nathan A. , Ramasamy Ravichandran , Wang Li Feng , Schaefer Saul
Published: December 2017

Nicotinic acid (niacin) has been shown to decrease myocyte injury. Because interventions that lower the cytosolic NADH/NAD+ratio improve glycolysis and limit infarct size, we hypothesized that 1) niacin, as a precursor of NAD+, would lower the NADH/NAD+ratio, increase glycolysis, and limit ischemic injury and 2) these cardioprotective benefits of niacin would be limited in conditions that block lactate removal. Isolated rat hearts were perfused without (Ctl) or with 1 μM niacin (Nia) and subjected to 30 min of low-flow ischemia (10% of baseline flow, LF) and reperfusion. To examine the effects of limiting lactate efflux, experiments were performed with 1) Ctl and Nia groups subjected to zero-flow ischemia and 2) the Nia group treated with the lactate-H+cotransport inhibitor α-cyano-4-hydroxycinnamate under LF conditions. Measured variables included ATP, pH, cardiac function, tissue lactate-to-pyruvate ratio (reflecting NADH/NAD+), lactate efflux rate, and creatine kinase release. The lactate-to-pyruvate ratio was reduced by more than twofold in Nia-LF hearts during baseline and ischemic conditions ( P < 0.001 and P< 0.01, respectively), with concurrent lower creatine kinase release than Ctl hearts ( P < 0.05). Nia-LF hearts had significantly greater lactate release during ischemia ( P < 0.05 vs. Ctl hearts) as well as higher functional recovery and a relative preservation of high-energy phosphates. Inhibiting lactate efflux with α-cyano-4-hydroxycinnamate and blocking lactate washout with zero flow negated some of the beneficial effects of niacin. During LF, niacin lowered the cytosolic redox state and increased lactate efflux, consistent with redox regulation of glycolysis. Niacin significantly improved functional and metabolic parameters under these conditions, providing additional rationale for use of niacin as a therapeutic agent in patients with ischemic heart disease.
Venous emptying mediates a transient vasodilation in the human forearm
Authors: Tschakovsky M. E. , Hughson R. L.
Published: December 2017

We tested the hypothesis that venous emptying serves as a stimulus for vasodilation in the human forearm. We compared the forearm blood flow (FBF; pulsed Doppler mean blood velocity and echo Doppler brachial artery diameter) response to temporary elevation of a resting forearm from below to above heart level when venous volume was allowed to drain versus when venous drainage was prevented by inflation of an upper arm cuff to ∼30 mmHg. Arm elevation resulted in a rapid reduction in venous volume and pressure. Cuff inflation just before elevation effectively prevented these changes. FBF was briefly reduced by ∼16% following arm elevation. A transient (86%) increase in blood flow began by ∼5 s of arm elevation and peaked by 8 s, indicating a vasodilation. This response was completely abolished by preventing venous emptying. Arterial inflow below heart level was markedly elevated by 343% following brief (4 s) forearm elevation. This hyperemia was minor when venous emptying during forearm elevation had been prevented. We conclude that venous emptying serves as a stimulus for a transient (within 10 s) vasodilation in vivo. This vasodilation can substantially elevate arterial inflow.
In vivo mechanisms of acetylcholine-induced vasodilation in rat sciatic nerve
Authors: Thomsen Kirsten , Rubin Inger , Lauritzen Martin
Published: December 2017

We examined the importance of nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF), and neurogenic activity in agonist-induced vasodilation and baseline blood flow [i.e., nerve microvascular conductance (NMVC)] in rat sciatic nerve using laser Doppler flowmetry. Agonists were acetylcholine (ACh) and 3-morpholinosydnonimine (SIN-1). Vasodilation occurring despite NO synthase (NOS) and cyclooxygenase inhibition and showing dependence on K+ channel activity was taken as being mediated by EDHF. NOS and cyclooxygenase inhibition with N ω-nitro-l-arginine (l-NNA) + indomethacin (Indo) revealed two phases of ACh-induced vasodilation: an initial, transient l-NNA + Indo-resistant vasodilation, peaking at 23 ± 6 s and lasting 145 ± 69 s, followed by sustainedl-NNA + Indo-sensitive vasodilation. l-NNA alone did not affect sustained ACh-induced vasodilation but decreased baseline NMVC by 55%. In the presence of l-NNA + Indo, the K+ channel blocker tetraethylammonium (TEA) inhibited transient ACh-induced vasodilation by 58% and reduced baseline NMVC by 25%. SIN-1-induced vasodilation increased fourfold in the presence of l-NNA, whereas the specific guanylyl cyclase inhibitor 1 H-(1, 2,4)oxadiazolo(4,3-α)quinoxalin-1-one abolished it. However, in homogenates of rat sciatic nerve, SIN-1-stimulated soluble guanylyl cyclase (sGC) activity was unaffected by l-NNA. TTX affected neither SIN-1- nor ACh-induced vasodilation. In conclusion, ACh-induced vasodilation consisted of two components, the first partially mediated by EDHF and the second by a vasodilatory prostanoid + NO. Baseline NMVC was dependent on NO and EDHF. Althoughl-NNA enhanced SIN-1-induced vasodilation, it had no effect on sGC-activity.
Effect of NO, phenylephrine, and hypoxemia on ductus venosus diameter in fetal sheep
Authors: Kiserud Torvid , Ozaki Takashi , Nishina Hidenori , Rodeck Charles , Hanson Mark A.
Published: December 2017

To study the regulation of the ductus venosus (DV) inlet in vivo, we measured the effect of vasoactive substances and hypoxemia on its diameter in nine fetal sheep in utero at 0.9 gestation under ketamine-diazepam anesthesia. Catheters were inserted into an umbilical vein and a fetal common carotid artery, and a flowmeter was placed around the umbilical veins. Ultrasound measurements of the diameter of the fetal DV during normoxic baseline conditions [fetal arterial Po2(Pao2) 24 mmHg] were compared with measurements during infusion of sodium nitroprusside (SNP; 1.3, 2.6, and 6.5 μg · kg−1· min−1) or the α1-adrenergic agonist phenylephrine (6.5 μg · kg−1· min−1) into the umbilical vein or during hypoxemia (fetal PaO2reduced to 10 mmHg). SNP increased the DV inlet diameter by 23%, but phenylephrine had no effect. Hypoxemia caused a 61% increase of the inlet diameter and a distension of the entire vessel. We conclude that the DV inlet is tonically constricted, because nitric oxide dilates it but an α1-adrenergic agonist does not potentiate constriction. Hypoxemia causes a marked distension of the entire DV.
Inotropic, chronotropic, and dromotropic effects mediated via parasympathetic ganglia in the dog heart
Authors: Tsuboi Masato , Furukawa Yasuyuki , Nakajima Koichi , Kurogouchi Fumio , Chiba Shigetoshi
Published: December 2017

Some parasympathetic ganglionic cells are located in the epicardial fat pad between the medial superior vena cava and the aortic root (SVC-Ao fat pad) of the dog. We investigated whether the ganglionic cells in the SVC-Ao fat pad control the right atrial contractile force, sinus cycle length (SCL), and atrioventricular (AV) conduction in the autonomically decentralized heart of the anesthetized dog. Stimulation of both sides of the cervical vagal complexes (CVS) decreased right atrial contractile force, increased SCL, and prolonged AV interval. Stimulation of the rate-related parasympathetic nerves to the sinoatrial (SA) node (SAPS) increased SCL and decreased atrial contractile force. Stimulation of the AV conduction-related parasympathetic nerves to the AV node prolonged AV interval. Trimethaphan, a ganglionic nicotinic receptor blocker, injected into the SVC-Ao fat pad attenuated the negative inotropic, chronotropic, and dromotropic responses to CVS by 33∼37%. On the other hand, lidocaine, a sodium channel blocker, injected into the SVC-Ao fat pad almost totally inhibited the inotropic and chronotropic responses to CVS and partly inhibited the dromotropic one. Lidocaine or trimethaphan injected into the SAPS locus abolished the inotropic responses to SAPS, but it partly attenuated those to CVS, although these treatments abolished the chronotropic responses to SAPS or CVS. These results suggest that parasympathetic ganglionic cells in the SVC-Ao fat pad, differing from those in SA and AV fat pads, nonselectively control the atrial contractile force, SCL, and AV conduction partially in the dog heart.
Liposome-induced pulmonary hypertension: properties and mechanism of a complement-mediated pseudoallergic reaction
Authors: Szebeni Janos , Baranyi Lajos , Savay Sandor , Bodo Mihaly , Morse David S. , Basta Milan , Stahl Gregory L. , Bünger Rolf , Alving Carl R.
Published: December 2017

Intravenous injection of liposomes can cause significant pulmonary hypertension in pigs, a vasoconstrictive response that provides a sensitive model for the cardiopulmonary distress in humans caused by some liposomal drugs. The reaction was recently shown to be a manifestation of “complement activation-related pseudoallergy” (CARPA; Szebeni J, Fontana JL, Wassef NM, Mongan PD, Morse DS, Dobbins DE, Stahl GL, Bünger R, and Alving CR. Circulation 99: 2302–2309, 1999). In the present study we demonstrate that the composition, size, and administration method of liposomes have significant influence on pulmonary vasoactivity, which varied between instantaneously lethal (following bolus injection of 5 mg lipid) to nondetectable (despite infusion of a 2,000-fold higher dose). Experimental conditions augmenting the pulmonary hypertensive response included the presence of dimyristoyl phosphatidylglycerol, 71 mol% cholesterol, distearoyl phosphatidylcholine, and hemoglobin in liposomes, increased vesicle size and polydispersity, and bolus injection vs. slow infusion. The vasoactivity of large multilamellar liposomes was reproduced with human C3a, C5a, and xenoreactive immunoglobulins, and it correlated with the complement activating and natural antibody binding potential of vesicles. Unilamellar, monodisperse liposomes with 0.19 ± 0.10 μm mean diameter had no significant vasoactivity. These data indicate that liposome-induced pulmonary hypertension in pigs is multifactorial, it is due to natural antibody-triggered classic pathway complement activation and it can be prevented by appropriate tailoring of the structure and administration method of vesicles.
Lowering of interstitial fluid pressure after neurogenic inflammation is inhibited by mystixin-7 peptide
Authors: Gjerde Eli-Anne B. , Woie Kathrine , Wei Edward T. , Reed Rolf K.
Published: December 2017

Soft tissue injury is accompanied by lowering of interstitial fluid pressure (Pif), plasma protein extravasation, and edema. Inflammation was produced by electrical stimulation (ES) of the vagus and the effects of the synthetic peptide mystixin-7 ( p-anisoyl-Arg-Lys-Leu-Leu-d-Thi-Ile-d-Leu-NH2) on Pif were examined. Micropuncture measurement of Pif in submucosa, without opening the trachea, was conducted on rats anesthetized with pentobarbital sodium (50 mg/kg) and euthanized with intravenous KCl. Pif in control (intravenous saline) was −1.2 ± 0.7 mmHg before ES and decreased to −4.7 ± 1.0 mmHg ( P < 0.01, n= 8) after ES. Mystixin-7 (10 and 20 μg/kg iv) blocked the fall in Pif after ES (−1.1 ± 0.3 and −0.8 ± 0.2 mmHg, P < 0.01, n = 8 and n= 4). The 1 μg/kg dose was without effect. When trachea from animals pretreated with mystixin-7 (20 μg/kg iv) were soaked in phosphate-buffered saline (0.15 M, pH 7.4), the rate of fluid accumulation was significantly reduced. This study suggests that mystixin peptides, which have structural similarity to a fragment from laminin-α1 chain, may be useful tools for studying cell adhesion and factors that maintain the structural integrity of connective tissue after injury.
Nitric oxide inhibits Ca2+mobilization through cADP-ribose signaling in coronary arterial smooth muscle cells
Authors: Yu Jiang-Zhou , Zhang David X. , Zou Ai-Ping , Campbell William B. , Li Pin-Lan
Published: December 2017

The present study was designed to determine whether the cADP-ribose-mediated Ca2+signaling is involved in the inhibitory effect of nitric oxide (NO) on intracellular Ca2+mobilization. With the use of fluorescent microscopic spectrometry, cADP-ribose-induced Ca2+release from sarcoplasmic reticulum (SR) of bovine coronary arterial smooth muscle cells (CASMCs) was determined. In the α-toxin-permeabilized primary cultures of CASMCs, cADP-ribose (5 μM) produced a rapid Ca2+release, which was completely blocked by pretreatment of cells with the cADP-ribose antagonist 8-bromo-cADP-ribose (8-Br-cADPR). In intact fura 2-loaded CASMCs, 80 mM KCl was added to depolarize the cells and increase intracellular Ca2+concentration ([Ca2+]i). Sodium nitroprusside (SNP), an NO donor, produced a concentration-dependent inhibition of the KCl-induced increase in [Ca2+]i, but it had no effect on the U-46619-induced increase in [Ca2+]i. In the presence of 8-Br-cADPR (100 μM) and ryanodine (10 μM), the inhibitory effect of SNP was markedly attenuated. HPLC analyses showed that CASMCs expressed the ADP-ribosyl cyclase activity, and SNP (1–100 μM) significantly reduced the ADP-ribosyl cyclase activity in a concentration-dependent manner. The effect of SNP was completely blocked by addition of 10 μM oxygenated hemoglobin. We conclude that ADP-ribosyl cyclase is present in CASMCs, and NO may decrease [Ca2+]iby inhibition of cADP-ribose-induced Ca2+mobilization.
Chronic metabolic sequelae of traumatic brain injury: prolonged suppression of somatosensory activation
Authors: Passineau M. J. , Zhao W. , Busto R. , Dietrich W. D. , Alonso O. , Loor J. Y. , Bramlett H. M. , Ginsberg M. D.
Published: December 2017

Injuries to the brain acutely disrupt normal metabolic function and may deactivate functional circuits. It is unknown whether these metabolic abnormalities improve over time. We used 2-deoxyglucose (2-DG) autoradiographic image-averaging to assess local cerebral glucose utilization (lCMRGlc) of the rat brain 2 mo after moderate (1.7–2.1 atm) fluid-percussion traumatic brain injury (FPI). Four animal groups ( n = 5 each) were studied: sham-injured rats with and without stimulation of the vibrissae-barrel field ipsilateral to injury; and animals with prior FPI, with or without this stimulation. In sham-injured rats, resting lCMRGlc was normal, and vibrissae stimulation produced right-sided metabolic activation of the ventrolateral thalamic and somatosensory-cortical projection areas. In rats with prior injury, lCMRGlccontralateral to injury was normal, but lCMRGlc of the ipsilateral forebrain was depressed by ∼38–45% compared with shams. Whisker stimulation in rats with prior trauma failed to induce metabolic activation of either cortex or thalamus. Image-mapping of histological material obtained in the same injury model was undertaken to assess the possible influence of injury-induced regional brain atrophy on computed lCMRGlc; an effect was found only in the lateral cortex at the trauma epicenter. Our results show that, 2 mo after trauma, resting cerebral metabolic perturbations persist, and the whisker-barrel somatosensory circuit shows no signs of functional recovery.
Preservation of myocardial function after adenoviral gene transfer in isolated myocardium
Authors: Lehnart S. E. , Janssen P. M. L. , Franz W. M. , Donahue J. K. , Lawrence J. H. , Marbán E. , Prestle J. , Hasenfuss G.
Published: December 2017

Adenoviral gene transfer to the heart represents a promising model for structure-function analyses. Rabbit hearts were subjected to an ex vivo perfusion protocol that achieves gene transfer in >90% of cardiac myocytes. Contractile function of isolated myocardial preparations of these hearts was then observed for 2 days in a recently developed trabecula culture system. In sham-infected hearts, the initial developed force (Finit) (15.6 ± 3.7 mN/mm2; n = 12) did not change significantly after 48 h (17.0 ± 1.9 mN/mm2; P = 0.46). In adenovirus-infected preparations, Finit (14.3 ± 1.8 mN/mm2; n = 21) did not significantly differ from the control ( P = 0.75) and was unchanged after 48 h (15.3 ± 2.5 mN/mm2; P= 0.93). After 2 days of continuous contractions, we observed homogenous and high-level expression of the reporter genes LacZ coding for β-galactosidase and Luc coding for firefly luciferase. Luciferase activity increased more than 2,500-fold from background levels of 8.7 × 103 ± 5.0 × 103 relative light units (RLU)/mg protein (from hearts transfected with promotorless adenovirus with luciferase transgene construct AdNULLLuc, n = 5) to 23.4 × 106 ± 11.1 × 106 RLU/mg protein (from hearts tranfected with adenovirus with Rous sarcoma virus promotor and luciferase transgene construct AdRSVLuc, n = 5) in infected myocardial preparations ( P < 0.005). Our results demonstrate a new ex vivo approach to achieve homogenous and high-level expression of recombinant adenoviral genes in contracting myocardium without adverse functional effects.
Effects of chronic exercise on calcium signaling in rat vascular endothelium
Authors: Chu Tzu-Fang , Huang Tung-Yi , Jen Chauying J. , Chen Hsiun-Ing
Published: December 2017

Chronic exercise enhances endothelium-dependent vasodilating responses. To investigate whether this is due to a change in endothelial Ca2+signaling, we examined intracellular Ca2+concentration ([Ca2+]i) level in rat aortic endothelium in response to acetylcholine (ACh) or ATP. Four-week-old male Wistar rats were divided into control and exercise groups. The exercised animals ran on a treadmill at a moderate intensity for 60 min/day, 5 day/wk, for 10 wk. Rat aortas were then excised and loaded with fura 2. After the aortas were mounted on a flow chamber, these specimens were observed under an epifluorescence microscope equipped with ratio-imaging capability. Our results showed that 1) chronic exercise increased both ACh- and ATP-induced [Ca2+]iresponses; 2) ACh induced heterogeneous [Ca2+]ielevation in individual endothelial cells; and 3) the exercise effect on ACh-evoked endothelial [Ca2+]ielevation was inhibited by the Ca2+influx blocker SKF-96365, by a Ca2+-free buffer, or by high concentrations of extracellular K+. We conclude that chronic exercise increases ACh-induced [Ca2+]ielevation in rat aortic endothelium in situ, possibly by facilitating Ca2+influx.
C-peptide exerts cardioprotective effects in myocardial ischemia-reperfusion
Authors: Young Lindon H. , Ikeda Yasuhiko , Scalia Rosario , Lefer Allan M.
Published: December 2017

Ischemia followed by reperfusion in the presence of polymorphonuclear leukocytes (PMNs) results in cardiac dysfunction. C-peptide, a cleavage product of proinsulin to insulin processing, induces nitric oxide (NO)-mediated vasodilation. NO is reported to attenuate cardiac dysfunction caused by PMNs after ischemia-reperfusion (I/R). Therefore, we hypothesized that C-peptide could attenuate PMN-induced cardiac dysfunction. We examined the effects of C-peptide in isolated ischemic (20 min) and reperfused (45 min) rat hearts perfused with PMNs. C-peptide (70 nmol/kg iv) given 4 or 24 h before I/R significantly improved coronary flow ( P < 0.05), left ventricular developed pressure (LVDP) ( P < 0.01), and the maximal rate of development of LVDP (+dP/d t max) compared with I/R hearts obtained from rats given 0.9% NaCl ( P < 0.01). N G-nitro-l-arginine methyl ester (l-NAME) (50 μmol/l) blocked these cardioprotective effects. In addition, C-peptide significantly reduced cardiac PMN infiltration from 183 ± 24 PMNs/mm2 in untreated hearts to 44 ± 10 and 58 ± 25 PMNs/mm2 in hearts from 4- and 24-h C-peptide-treated rats, respectively. Rat PMN adherence to rat superior mesenteric artery exposed to 2 U/ml thrombin was significantly reduced in rats given C-peptide compared with rats given 0.9% NaCl ( P < 0.001). Moreover, C-peptide enhanced basal NO release from rat aortic segments. These results provide evidence that C-peptide can significantly attenuate PMN-induced cardiac contractile dysfunction in the isolated perfused rat heart subjected to I/R at least in part via enhanced NO release.