bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitric oxide triggers programmed cell death (apoptosis) of adult rat ventricular myocytes in culture
Authors: Pinsky David J. , Aji Walif , Szabolcs Matthias , Athan Eleni S. , Liu Youping , Yang Yi Ming , Kline Richard P. , Olson Kim E. , Cannon Paul J.
Published: December 2017

Excessive nitric oxide (NO) production within the heart is implicated in the pathogenesis of myocyte death, but the mechanism whereby NO kills cardiac myocytes is not known. To determine whether NO may trigger programmed cell death (apoptosis) of adult rat ventricular myocytes in culture, the NO donor S-nitroso- N-acetylpenicillamine (SNAP) was shown to kill purified cardiac myocytes in a dose-dependent fashion. In situ analysis of ventricular myocytes plated on chamber slides using nick-end labeling of DNA demonstrated that SNAP induces cardiac myocyte apoptosis, which was confirmed by the identification of oligonucleosomal DNA fragmentation on agarose gel electrophoresis. Similarly, treatment of cardiac myocytes with cytokines that induce inducible NO synthase was shown to cause an NO-dependent induction of apoptosis. Addition of reduced hemoglobin to scavenge NO liberated by SNAP extinguished both the increase in percentage of apoptotic cells and the appearance of DNA ladders. Treatment with SNAP (but not with N-acetylpenicillamine or SNAP + hemoglobin) not only induced apoptosis but resulted in a marked increase in p53 expression in cardiac myocytes detected by Western blotting and immunohistochemistry. These data indicate that NO has the capacity to kill cardiac myocytes by triggering apoptosis and suggest the involvement of p53 in this process.
Transport of fluid and solutes in the body I. Formulation of a mathematical model
Authors: Gyenge C. C. , Bowen B. D. , Reed R. K. , Bert J. L.
Published: December 2017

A compartmental model of short-term whole body fluid, protein, and ion distribution and transport is formulated. The model comprises four compartments: a vascular and an interstitial compartment, each with an embedded cellular compartment. The present paper discusses the assumptions on which the model is based and describes the equations that make up the model. Fluid and protein transport parameters from a previously validated model as well as ionic exchange parameters from the literature or from statistical estimation [see companion paper: C. C. Gyenge, B. D. Bowen, R. K. Reed, and J. L. Bert. Am. J. Physiol. 277 ( Heart Circ. Physiol. 46): H1228–H1240, 1999] are used in formulating the model. The dynamic model has the ability to simulate 1) transport across the capillary membrane of fluid, proteins, and small ions and their distribution between the vascular and interstitial compartments; 2) the changes in extracellular osmolarity; 3) the distribution and transport of water and ions associated with each of the cellular compartments; 4) the cellular transmembrane potential; and 5) the changes of volume in the four fluid compartments. The validation and testing of the proposed model against available experimental data are presented in the companion paper.
Reversal of hypertension by angiotensin II type 1 receptor antisense gene therapy in the adult SHR
Authors: Katovich Michael J. , Gelband Craig H. , Reaves Phyllis , Wang Hong-Wei , Raizada Mohan K.
Published: December 2017

Pharmacological blockade of the renin-angiotensin system in both hypertensive patients and animal models such as the spontaneously hypertensive rat (SHR) effectively reduces blood pressure (BP). Recent studies have established that virally mediated delivery (vector LNSV) of antisense to the angiotensin II type 1 receptor (LNSV-AT1R-AS) will attenuate or abolish the development of hypertension in the SHR. However, the effectiveness of this gene therapy approach to reduce high BP once it is established in the adult has not been ascertained. In this study, we investigated the hypothesis that viral delivery of AT1R-AS into the adult SHR will reduce BP and reverse the vascular reactivity associated with the hypertension. Intracardiac injection of virus particles containing LNSV-AT1R-AS into adult SHR resulted in a 30- to 60-mmHg reduction in BP that was maintained for up to 36 days compared with SHR treated with virus alone (LNSV without antisense). Measurement of renal resistance arteriolar reactivity demonstrated a leftward shift in the KCl and phenylephrine concentration-response relationships and an impaired endothelium-dependent relaxation to ACh in LNSV-treated SHR compared with control Wistar-Kyoto rats. These vascular alterations were reversed in the LNSV-AT1R-AS-treated SHR. Collectively, these data demonstrate that virally mediated gene delivery of AT1R-AS can effectively reduce BP and reverse renovascular pathophysiology associated with the hypertensive state when administered to the adult SHR.
Neuropeptide Y contributes to innervation-dependent increase inICa,Lvia ventricular Y2receptors
Authors: Protas Lev , Robinson Richard B.
Published: December 2017

The developmental increase in L-type Ca current ( ICa,L) density in the rat ventricle is reproduced in vitro by culturing neonatal myocytes with sympathetic neurons. We tested whether this effect of sympathetic innervation results from a chronic or sustained action of neurally released neuropeptide Y (NPY). Ventricular myocytes from newborn rats were cultured in serum-free medium with or without sympathetic neurons, NPY, or NPY analogs. Ca currents were measured in single myocytes at room temperature using the perforated patch clamp. In all cell groups (control, innervated, or NPY treated), the current-voltage relation for ICa,Lwas represented by a bell-shaped curve with maximal value near 0 mV. The current density at 0 mV normalized to that of corresponding mean control values was 1.63 ± 0.12 and 1.52 ± 0.16 for innervated and NPY-treated myocytes, respectively. Both groups differed significantly from control ( P < 0.05). NPY analogs exhibited the following rank order of effectiveness: NPY ≥ NPY-(13—36) ≥ PYY >> [Leu31Pro34]NPY, suggesting that the NPY effect occurs via a Y2-receptor subtype. In confirmation, chronic treatment of innervated cultures with a Y2-selective NPY antagonist prevented the innervation-dependent increase in ICa,L. These results indicate that sympathetic innervation contributes to the developmental increase in ICa,Lvia neurally released NPY acting at Y2receptors on the ventricular myocytes.
NO from smooth muscle cells decreases NOS expression in endothelial cells: role of TNF-α
Authors: de Frutos Trinidad , de Miguel Lourdes Sánchez , García-Durán Margarita , González-Fernández Fernando , Rodríguez-Feo Juan A. , Montón Mercedes , Guerra José , Farré Jerónimo , Casado Santos , López-Farré Antonio
Published: December 2017

Despite the evidence that cytokines stimulate nitric oxide (NO) production by inducible nitric oxide synthase (iNOS), several reports recently demonstrated that the hypotensive response related to endothelial nitric oxide synthase (eNOS) activity could be inhibited by the same cytokines. The aim of the present work was to analyze whether NO generated by vascular smooth muscle cells (VSMC) could modify eNOS protein expression in endothelial cells. Bovine aortic endothelial cells (BAEC) and bovine VSMC (BVSMC) in coculture were used for the study. Interleukin-1β (IL-1β, 10 ng/ml)-treated BVSMC, which expressed iNOS protein, decreased eNOS protein expression in BAEC. The presence of NO antagonists Nω-nitro-l-arginine methyl ester (10−3mol/l) or NG-monomethyl-l-arginine (10−3mol/l) prevented the decrease in eNOS protein expression induced by IL-1β-treated BVSMC. Surprisingly, two different NO donors, 3-morpholinosydnonimine (10−4mol/l) and S-nitroso- N-acetyl-d,l-penicillamine (10−4mol/l), failed to modify eNOS expression in BAEC, suggesting the existence of a diffusible mediator released from IL-1β-treated BVSMC that acts on endothelial cells by reducing eNOS expression. The presence of NO antagonists reduced tumor necrosis factor-α (TNF-α) production by IL-1β-stimulated BVSMC. This effect was also produced in the presence of a protein kinase G inhibitor, guanosine-5′-O-(2-thiodiphosphate) trilithium salt. A polyclonal antibody against TNF-α prevented eNOS expression in the BAEC-BVSMC coculture. In conclusion, NO by itself failed to modify eNOS protein expression in endothelial cells but increased TNF-α generation by IL-1β-stimulated BVSMC and, in this way, reduced eNOS expression in the endothelium.
A presynaptic mechanism contributes to depression of autonomic signal transmission in NTS
Authors: Chen Chao-Yin , Horowitz John M. , Bonham Ann C.
Published: December 2017

With increasing frequencies of autonomic afferent input to the nucleus tractus solitarii (NTS), postsynaptic responses are depressed. To test the hypothesis that a presynaptic mechanism contributes to this frequency-dependent depression, we used whole cell, voltage-clamp recordings in an NTS slice. First, we determined whether solitary tract stimulation (0.4–24 Hz) resulted in frequency-dependent depression of excitatory postsynaptic currents (EPSCs) in second-order neurons. Second, because decreases in presynaptic glutamate release result in a parallel depression of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and N-methyl-d-aspartic acid (NMDA) receptor-mediated components of EPSCs, we determined whether the magnitude, time course, and recovery from the depression were the same in both EPSC components. Third, to determine whether AMPA receptor desensitization contributed, we examined the depression during cyclothiazide. EPSCs decreased in a frequency-dependent manner by up to 76% in second- and 92% in higher-order neurons. AMPA and NMDA EPSC components were depressed with the same magnitude (by 83% and 83%) and time constant (113 and 103 ms). The time constant for the recovery was also not different (1.2 and 0.8 s). Cyclothiazide did not affect synaptic depression at ≥3 Hz. The data suggest that presynaptic mechanism(s) at the first NTS synapse mediate frequency-dependent synaptic depression.
NO mediates postjunctional inhibitory effect of neurogenic ACh in guinea pig small intestinal microcirculation
Authors: Kotecha N. , Coffa F. P.
Published: December 2017

The present study was designed to evaluate the role of the endothelium as an effector organ of neurally mediated inhibition of vascular tone. Acetylcholine (ACh), either released by stimulation of the submucosal ganglia or applied exogenously, inhibited phenylephrine (PE)-induced constrictions in arterioles of the guinea pig intestinal submucosa. N G-monomethyl-l-arginine (l-NMMA), an inhibitor of nitric oxide (NO) synthesis, attenuated the response to superfused ACh by 74% compared with 94% attenuation obtained with N G-nitro-l-arginine (l-NNA).l-NNA attenuated the response to neurally released ACh by 98% and that to iontophoretically applied ACh by 92%. l-Arginine reversed the effects of both l-NMMA andl-NNA. Functional integrity of the endothelium was essential for the neurally mediated inhibition of PE-induced constrictions. However, neurogenic inhibition of neurally evoked constrictions was preserved despite endothelial disruption. It was concluded that at the postjunctional level, the mechanism of action of neurally released ACh was almost exclusively via a NO-dependent pathway, with the source of NO being the vascular endothelium.
Cerebrovascular reactivity to CO2 and hypotension after mild cortical impact injury
Authors: Golding Elke M. , Steenberg Marie L. , Contant Charles F. , Krishnappa Indra , Robertson Claudia S. , Bryan Robert M.
Published: December 2017

Cerebrovascular reactivity to CO2 or hypotension was studied in vivo and in vitro [pressurized arteries (∼82 μm) and arterioles (∼30 μm)] at 1 h after mild controlled cortical impact (CCI) injury in rats. The cortical perfusion response [assessed using laser-Doppler flowmetry (LDF)] to altered CO2 was diminished (up to 81%) after mild CCI injury. The responses to CO2 alterations in arteries and arterioles isolated from the injured cortex were similar to responses in vessels isolated from sham-injured animals. After mild CCI injury, the autoregulatory response to hypotension (measured using LDF) was maintained or even enhanced, depending on the method used to measure the response. Vessels isolated from the injury site showed a response to changes in pressure similar to that in vessels isolated from sham-injured rats. We conclude that mild CCI injury produces complicated alterations in cerebrovascular control. Whereas the autoregulatory response to hypotension was maintained or even enhanced, the in vivo vascular response to CO2 was severely compromised. The altered response to CO2 was not caused by an intrinsic vascular perturbation but rather an altered milieu after mild CCI injury.
Ca2+-activated Cl− current can be triggered by Na+ current-induced SR Ca2+ release in rabbit ventricle
Authors: Sun Hui , Chartier Denis , Nattel Stanley , Leblanc Normand
Published: December 2017

The Ca2+-activated Cl− current [ I Cl(Ca)] contributes to the repolarization of the cardiac action potential under physiological conditions. I Cl(Ca) is known to be primarily activated by Ca2+release from the sarcoplasmic reticulum (SR). L-type Ca2+ current [ I Ca(L)] represents the major trigger for Ca2+ release in the heart. Recent evidence, however, suggests that Ca2+ entry via reverse-mode Na+/Ca2+exchange promoted by voltage and/or Na+ current ( I Na) may also play a role. The purpose of this study was to test the hypothesis that I Cl(Ca) can be induced by I Na in the absence of I Ca(L). Macroscopic currents and Ca2+transients were measured using the whole cell patch-clamp technique in rabbit ventricular myocytes loaded with Indo-1. Nicardipine (10 μM) abolished I Ca(L)at a holding potential of −75 mV as tested in Na+-free external solution. In the presence of 131 mM external Na+and in the absence of I Ca(L), a 4-aminopyridine-resistant transient outward current was recorded in 64 of 81 cells accompanying a phasic Ca2+ transient. The current reversed at −42.0 ± 1.3 mV ( n = 6) and at +0.3 ± 1.4 mV ( n = 6) with 21 and 141 mM of internal Cl−, respectively, similar to the predicted reversal potential with low intracellular Cl− concentration ([Cl−]i) (−47.8 mV) and high [Cl−]i(−1.2 mV). Niflumic acid (100 μM) inhibited the current without affecting the Ca2+ signal ( n = 8). Both the current and Ca2+ transient were abolished by 10 mM caffeine ( n = 6), 10 μM ryanodine ( n = 3), 30 μM tetrodotoxin ( n = 9), or removal of extracellular Ca2+( n = 6). These properties are consistent with those of I Cl(Ca)previously described in mammalian cardiac myocytes. We conclude that 1) I Cl(Ca) can be recorded in the absence of I Ca(L), and 2) I Na-induced SR Ca2+ release mechanism is also present in the rabbit heart and may play a physiological role in activating the Ca2+-sensitive membrane Cl− conductance.
Origins of heart rate variability: relationship of heart rate burst morphology to work duration and load
Authors: Roach Daniel , Haennel Robert , Koshman Mary Lou , Sheldon Robert
Published: December 2017

We are developing a lexicon of specific heart period changes, or lexons, that recur frequently and whose physiological meaning can be read into ambulatory electrocardiogram (ECG). The transient, reversible “burst” of tachycardia induced by exercise initiation can also be seen on ambulatory ECG. We hypothesized that burst morphology depended on the work that preceded it and on baroreceptor activation. Ten subjects with mean age 38 yr (range 17–69 yr) underwent two protocols of semisupine cycling in which load and duration were varied. Burst duration increased with longer cycling times (median values of 18.0, 25.5, and 23.7 s with 1, 3, and 5 s of cycling, respectively; P= 0.033). Burst shape as assessed by heart period exponential decay constant and burst magnitude did not change. To assess the impact of workload, subjects cycled for 5 s at loads of 0, 25, 50, and 75 W. No significant differences were seen in burst duration, burst magnitude, or burst shape. Tachycardia preceded hypotension by 4.6 ± 2.2 s, which is inconsistent with baroreceptor involvement in the onset of burst tachycardia. Because burst morphology is a nearly quantal response to the initiation of exercise, the presence of a burst on an ambulatory ECG implies the onset of exercise.
Further attenuation of endothelium-dependent relaxation imparted by natriuretic peptide receptor antagonism
Authors: Wennberg Paul W. , Miller Virginia M. , Rabelink Ton , Burnett John C.
Published: December 2017

Nitric oxide (NO) is an important endothelium-derived relaxing factor that functions via activation of soluble guanylyl cyclase and cGMP generation in vascular smooth muscle. Recently, studies have described the synthesis and secretion of C-type natriuretic peptide (CNP) from endothelial cells. This peptide also mediates relaxation via cGMP but through activation of particulate guanylyl cyclase. We tested the hypothesis that endothelium-dependent relaxations to acetylcholine or bradykinin in isolated canine coronary arteries involve both releases of NO and CNP. Rings of canine coronary arteries were incubated with either inhibitors of NO production ( NG-monomethyl-l-arginine,l-NMMA) or the natriuretic peptide receptor antagonist HS-142-1. CNP caused concentration-dependent relaxations of rings with and without endothelium. These relaxations were attenuated by HS-142-1. Relaxations to acetylcholine and bradykinin were attenuated byl-NMMA alone but not attenuated by HS-142-1 alone. Coinhibition withl-NMMA and HS-142-1 significantly inhibited acetylcholine- and bradykinin-induced relaxation to a magnitude greater than either inhibitor alone. In summary, a novel interaction between the NO and the natriuretic peptide system is demonstrated by increased attenuation of endothelium-dependent relaxations to acetylcholine and bradykinin when both NO synthase and natriuretic peptide receptors are inhibited. These investigations support the concept of release of multiple endothelium-derived factors in response to acetylcholine- and bradykinin-receptor stimulation in endothelial cells, which may include CNP, as well as NO.
Evidence for peroxynitrite as a signaling molecule in flow-dependent activation of c-Jun NH2-terminal kinase
Authors: Go Young-Mi , Patel Rakesh P. , Maland Matthew C. , Park Heonyong , Beckman Joseph S. , Darley-Usmar Victor M. , Jo Hanjoong
Published: December 2017

The c-Jun NH2-terminal kinase (JNK), also known as stress-activated protein kinase, is a mitogen-activated protein kinase that determines cell survival in response to environmental stress. Activation of JNK involves redox-sensitive mechanisms and physiological stimuli such as shear stress, the dragging force generated by blood flow over the endothelium. Laminar shear stress has antiatherogenic properties and controls structure and function of endothelial cells by mechanisms including production of nitric oxide (NO) and superoxide ([Formula: see text]). Here we show that both NO and [Formula: see text] are required for activation of JNK by shear stress in endothelial cells. The present study also demonstrates that exposure of endothelial cells to shear stress increases tyrosine nitration, a marker of reactive nitrogen species formation. Furthermore, inhibitors or scavengers of NO, [Formula: see text], or reactive nitrogen species prevented shear-dependent increase in tyrosine nitration and activation of JNK. Peroxynitrite alone, added to cells as a bolus or generated over 60 min by 3-morpholinosydnonimine, also activates JNK. These results suggest that reactive nitrogen species, in this case most likely peroxynitrite, act as signaling molecules in the mechanoactivation of JNK.
Dependence of Ca2+sensitivity of arterial contractions on history of receptor activation
Authors: Ratz Paul H.
Published: December 2017

Stimulation of receptors causing arterial contraction may also cause attenuation of cell responsiveness to stimuli. This study tested the hypothesis that attenuation of receptor-induced contractions involves Ca2+desensitization. Renal artery rings were pretreated with 10 μM phenylephrine (PE), relaxed with PE washout (plus phentolamine), and then activated by histamine (HA). Pretreatment for 30 min resulted in a rightward shift in the concentration-contraction curve to HA by ∼½ log without a reduction in the slope or maximum response. For example, control and PE-pretreated tissues responded to 0.56 μM HA with strong (0.95 F/ Fo) and weak (0.16 F/ Fo) contractions, respectively, where F/ Forepresents contractile force. This reduced reactivity was completely reversed within 90 min. In fura-loaded tissues, PE pretreatment caused less of a rightward shift in the HA concentration-intracellular free Ca2+concentration ([Ca2+]i) curve than in the HA concentration-contraction curve. A dissociation between force and [Ca2+]iwas also produced when KCl was used instead of HA. These data suggest that the reduced reactivity produced by PE pretreatment involved, in part, a reduction in the ability of HA to increase the Ca2+sensitivity of contractions. These data support the hypothesis that the degree of stimulus-induced Ca2+sensitization of contractions is dependent on the history of receptor activation.
Role of voltage-sensitive release mechanism in depression of cardiac contraction in myopathic hamsters
Authors: Howlett Susan E. , Xiong Wei , Mapplebeck Cindy L. , Ferrier Gregory R.
Published: December 2017

We investigated excitation-contraction (EC) coupling in isolated ventricular myocytes from prehypertrophic cardiomyopathic (CM) hamster hearts. Conventional and voltage-clamp recordings were made with high-resistance microelectrodes, and cell shortening was measured with a video-edge detector at 37°C. Contractions were depressed in myocytes from CM hearts, whether they were initiated by action potentials or voltage-clamp steps. As in guinea pig and rat, contraction in hamster myocytes could be triggered by a voltage-sensitive release mechanism (VSRM) or Ca2+-induced Ca2+ release (CICR). Selective activation of these mechanisms demonstrated that the defect in EC coupling was primarily caused by a defect in the VSRM. However, activation and inactivation properties of the VSRM were not altered. When the VSRM was inhibited, the remaining contractions induced by CICR exhibited identical bell-shaped contraction voltage relations in normal and CM myocytes. Inward Ca2+current was unchanged. Thus a defect in the VSRM component of EC coupling precedes the development of hypertrophy and failure in CM hamster heart.
AT1 receptor regulation in salt-sensitive hypertension
Authors: Strehlow Kerstin , Nickenig Georg , Roeling Jörg , Wassmann Sven , Zolk Oliver , Knorr Andreas , Böhm Michael
Published: December 2017

The molecular events governing salt-sensitive hypertension are currently unknown. Because the renin-ANG system plays a central role in blood pressure regulation and electrolyte balance, it may be closely involved in the phenomenon of salt sensitivity. Therefore, we examined the effect of a high-salt diet (8%) and a low-salt diet (0.4%) on ANG II-caused vascular constriction and ANG II type 1 (AT1) receptor expression in aorta, brain, and kidney of Dahl S (salt-sensitive) and Dahl R (salt-resistant) rats by means of radioligand binding assays and quantitative PCR. NaCl diet at 8% led to a significant increase of blood pressure in Dahl S but not in Dahl R rats. High-sodium intake caused a profound decrease of ANG II-induced aortic vasoconstriction in both Dahl R and Dahl S rats. The underlying mechanism was a downregulation of aortic AT1receptor density and AT1 receptor mRNA. AT1 receptor mRNA was downregulated to 57.8% in Dahl R and 59.0% in Dahl S rats by an 8% NaCl diet compared with a 0.4% NaCl diet ( P < 0.05). There was a similar decrease in aortic AT1 receptor density. Additionally, AT1receptor mRNA was also downregulated in the kidney but upregulated the brain of Dahl R and S rats on a high-salt diet. Thus high NaCl intake causes organ-specific AT1 receptor regulation in Dahl R and in Dahl S rats despite the differential blood pressure regulation in these animal models in response to a high-salt diet. These findings suggest that the regulation of vascular AT1 receptors is influenced by numerous factors such as the renin-ANG system and obviously by various other events that are currently only partly understood.
Downregulation of voltage-gated K+channels in rat heart with right ventricular hypertrophy
Authors: Lee Jong-Kook , Nishiyama Atsushi , Kambe Fukushi , Seo Hisao , Takeuchi Susumu , Kamiya Kaichiro , Kodama Itsuo , Toyama Junji
Published: December 2017

The effects of myocardial hypertrophy on mRNA expression levels of voltage-gated K+channels were investigated using monocrotaline (MCT)-induced pulmonary hypertensive rats. The ratio of right ventricle weight to left ventricle plus septum weight on day 28 was increased significantly compared with control rats [control vs. MCT: 0.27 ± 0.01 vs. 0.58 ± 0.03 ms ( n = 8–13); P < 0.05]. Electrocardiograms showed that QRS duration [control vs. MCT: 26.4 ± 2.6 ms vs. 31.5 ± 5.8 ms ( n = 6); P < 0.05], Q-T interval [control vs. MCT: 100.8 ± 8.9 ms vs. 110.0 ± 4.2 ms ( n = 6); P < 0.05] and corrected Q-T interval [Q-Tc; control vs. MCT: 8.4 ± 0.7 ms vs. 10.2 ± 0.4 ms ( n = 6); P < 0.05] were prolonged significantly on day 28. mRNA levels of Kv1.2, 1.5, 2.1, 4.2, and 4.3 for day 28 assessed by ribonuclease protection assays were decreased significantly from control by 60 ± 10, 76 ± 3, 58 ± 5, 81 ± 5, and 45 ± 12%, respectively ( n = 3; P < 0.005), and Kv1.4 mRNA level for day 28 was unaffected [Kv1.4, control vs. MCT: 1.0 ± 0.28 vs. 0.88 ± 0.44 (arbitrary units) ( n = 3); not significant (NS)]. On the other hand, there was no significant difference between control and MCT rats in mRNA levels of these Kv channels for day 14 [Kv1.2 (control vs. MCT): 1.0 ± 0.25 vs. 0.87 ± 0.18 ( n = 3), NS; Kv1.4: 1.0 ± 0.22 vs. 1.27 ± 0.37 ( n = 3), NS; Kv1.5: 1.0 ± 0.16 vs. 0.91 ± 0.28 ( n = 3), NS; Kv2.1: 1.0 ± 0.26 vs. 0.99 ± 0.25 ( n = 3), NS; Kv4.2: 1.0 ± 0.15 vs. 1.22 ± 0.28 ( n = 3), NS; Kv4.3: 1.0 ± 0.20 vs. 1.21 ± 0.28 ( n= 3), NS]. These findings suggest that altered ventricular repolarization at the advanced stage of hypertrophy may be the result of an inhibition of gene expression of multiple types of voltage-gated K+channels.
PKC-dependent activation of p46/p54 JNKs during ischemic preconditioning in conscious rabbits
Authors: Ping Peipei , Zhang Jun , Huang Shuang , Cao Xinan , Tang Xian-Liang , Li Richard C. X. , Zheng Yu-Ting , Qiu Yumin , Clerk Angela , Sugden Peter , Han Jiahuai , Bolli Roberto
Published: December 2017

A conscious rabbit model was used to study the effect of ischemic preconditioning (PC) on stress-activated kinases [c-Jun NH2-terminal kinases (JNKs) and p38 mitogen-activated protein kinase (MAPK)] in an environment free of surgical trauma and attending external stress. Ischemic PC (6 cycles of 4-min ischemia/4-min reperfusion) induced significant activation of protein kinase C (PKC)-ε in the particulate fraction, which was associated with activation of p46 JNK in the nuclear fraction and p54 JNK in the cytosolic fraction; all of these changes were completely abolised by the PKC inhibitor chelerythrine. Selective enhancement of PKC-ε activity in adult rabbit cardiac myocytes resulted in enhanced activity of p46/p54 JNKs, providing direct in vitro evidence that PKC-ε is coupled to both kinases. Studies in rabbits showed that the activation of p46 JNK occurred during ischemia, whereas that of p54 JNK occurred after reperfusion. A single 4-min period of ischemia induced a robust activation of the p38 MAPK cascade, which, however, was attenuated after 5 min of reperfusion and disappeared after six cycles of 4-min ischemia/reperfusion. Overexpression of PKC-ε in cardiac myocytes failed to increase the p38 MAPK activity. These results demonstrate that ischemic PC activates p46 and p54 JNKs via a PKC-ε-dependent signaling pathway and that there are important differences between p46 and p54 JNKs with respect to the subcellular compartment (cytosolic vs. nuclear) and the mechanism (ischemia vs. reperfusion) of their activation after ischemic PC.
Contribution of the Na+channel and Na+/H+exchanger to the anoxic rise of [Na+] in ventricular myocytes
Authors: Eigel B. N. , Hadley R. W.
Published: December 2017

The aim of this study was to quantify the contribution of the Na+/H+exchanger (NHE) and the Na+channel to the rise in cytosolic Na+concentration ([Na+]) that is seen in anoxic guinea pig ventricular myocytes. [Na+] was measured with the use of microfluorometry and was found to rise to 44 mM after prolonged anoxia. This rise was partially sensitive to either TTX or HOE-642, selective inhibitors of the Na+channel and NHE1, respectively. [Na+] did not significantly rise when both drugs were present, suggesting that other routes of Na+entry were insignificant. However, the relative contributions of the NHE and the Na+channel were found to be remarkably sensitive to ionic conditions expected to occur during ischemia. The Na+channel was the dominant Na+source during acidic anoxia. However, the NHE was the dominant Na+source during both hyperkalemic anoxia and simulated ischemia (hyperkalemia, low pH, and anoxia). The data suggest that the NHE may prove to be the best pharmacological target to reduce Na+entry during true ischemia and that inhibition of Na+influx could contribute strongly to the cardioprotective effects of NHE inhibitors.
Gsand adenylyl cyclase in transverse tubules of heart: implications for cAMP-dependent signaling
Authors: Laflamme Michael A. , Becker Peter L.
Published: December 2017

The transverse tubules are highly specialized invaginations of the cardiac sarcolemmal membrane involved in excitation-contraction (EC) coupling. Several proteins directly involved in EC coupling have been shown to reside either in the transverse tubular membrane or in closely associated structures. With the use of immunofluorescence microscopy, we have found that GSand adenylyl cyclase, key elements in the β-adrenergic signal transduction cascade, are essentially homogeneously distributed throughout the transverse tubular network of isolated rat ventricular myocytes. GS, in particular, was much more abundant within the transverse tubular membrane than in the peripheral sarcolemma. Furthermore, both proteins are also present in the intercalated disk region. The location of these elements of the cAMP-signaling cascade within a few micrometers of every inotropic target suggests that control and action of this second messenger are quite local. Furthermore, a similar distribution is likely for negatively inotropic receptor systems that oppose GS-linked receptors at the level of adenylyl cyclase. Thus, in addition to their role in EC coupling, transverse tubules appear to be the primary site for signaling by inotropic agents.
Role of endothelin-1 in age-dependent cerebrovascular hypotensive responses after brain injury
Authors: Armstead William M.
Published: December 2017

This study was designed to compare the effect of fluid percussion brain injury (FPI) on the hypotensive cerebrovascular response in newborn and juvenile pigs as a function of time postinsult and to determine the role of endothelin-1 (ET-1) in any age-dependent differences in hypotensive cerebrovascular regulation after injury. Ten minutes of hypotension (10–15 ml blood/kg) decreased mean arterial blood pressure uniformly in both groups (∼45%). In the newborn, hypotensive pial artery dilation (PAD) was blunted within 1 h, remained diminished for at least 72 h, but was resolved within 168 h postinjury (66 ± 4, 69 ± 4, 71 ± 4, and 64 ± 4% inhibition at 1, 4, 8, and 72 h post-FPI). During normotension, regional cerebral blood flow (rCBF) was decreased by FPI, and hypotension further reduced the already decremented rCBF for at least 72 h. Cerebrospinal fluid (CSF) ET-1 was increased from 26 ± 4 to 206 ± 25 pg/ml within 72 h post-FPI, whereas an ET-1 antagonist partially restored impaired hypotensive PAD and altered hypotensive rCBF. In contrast, hypotensive PAD and altered CBF were only inhibited for 4 h post-FPI in the juvenile (56 ± 3 and 34 ± 4% inhibition at 1 and 4 h post-FPI). CSF ET-1 was only increased from 27 ± 4 to 67 ± 9 pg/ml at 4 h, whereas the concentration returned to preinjury value by 8 h post-FPI. ET-1 antagonism similarly partially restored impaired hypotensive PAD and altered hypotensive rCBF. These data show that FPI disturbs cerebral autoregulation during hypotension both to a greater magnitude and for a longer duration in the newborn than in the juvenile. These data suggest that the greater FPI-induced ET-1 release in the newborn could contribute to age-dependent differences in impaired hypotensive cerebral autoregulation after FPI.
ACE inhibitors in HF restore canine pulmonary endothelial function and ANG II vasoconstriction
Authors: Straeter-Knowlen Ingrid M. , Dell'italia Louis J. , Dai Jun , Hankes Gerald H. , Dillon A. Raymond , Cartee R. Earl , Pohost Gerald M. , Ku David D.
Published: December 2017

Chronic mitral regurgitation (MR) in dogs results in pulmonary congestion and increased cardiac angiotensin-converting enzyme (ACE) activity and angiotensin (ANG) II levels. ACE could contribute to altered pulmonary vasomotion in heart failure, and ACE inhibitor (ACEI) therapy may normalize pulmonary vasomotion. We evaluated pulmonary artery (PA) responses to ANG II and bradykinin (BK) in control dogs, in dogs with 4 mo of MR, in MR dogs treated with the ACEI ramipril (MR + R), and in control dogs treated with ramipril (C + R). Mean PA systolic pressure increased in MR dogs (21 ± 4 mmHg) but was normal in MR + R dogs (13 ± 1 mmHg). Constriction of PA rings to ANG II was depressed in MR dogs. ACEI treatment (MR + R) restored ANG II responsiveness, but peak ANG II response (3.6 ± 0.2 g) in MR + R dogs remained lower than in C + R dogs (4.7 ± 0.2 g). Endothelium-dependent relaxation to BK was decreased (−87 ± 4% C, −65 ± 4% MR; P < 0.05). Ramipril (MR + R) restored relaxation to BK. This demonstrates that pulmonary congestion results in impaired pulmonary vasomotion to ANG II and BK, which ACEIs could normalize, supporting the use of ACEIs in clinical management of chronic congestive heart failure.
Cablin: a novel protein of the capillary basal lamina
Authors: Charron Audra J. , Xu Weimin , Bacallao Robert L. , Wandinger-Ness Angela
Published: December 2017

The microvascular wall is remarkably simple, consisting only of the endothelial lining, subjacent basal lamina, and underlying periendothelial cells. This study describes the characterization of a novel microvascular protein. This 80,000-molecular weight protein was predominantly associated with electron-lucent amorphous material in capillary basal laminae and therefore termed cablin (protein of the capillary basal lamina). Consistent with its immunolocalization to the microvasculature, cablin was synthesized and secreted by cultured endothelial cells and vascular smooth muscle cells. Furthermore, cablin expression was induced during neovascularization. The predicted amino acid sequence of cablin revealed a prevalence of polar amino acids. Accounting for the low yet significant homology to several α-helical proteins, these residues were best accommodated by secondary structure predictions that aligned the molecule into two large α-helical domains. The presence of the integrin-binding RGD tripeptide and a putative elastin-binding sequence suggest that this rodlike molecule is suited to cross-link cells and matrix constituents. In this capacity it could contribute to the mechanical strength or the angiogenic potential of the microvasculature.
Oxidized low-density lipoprotein induces cytoskeletal disorganization in smooth muscle cells
Authors: Massaeli Hamid , Hurtado Cecilia , Austria J. Alejandro , Pierce Grant N.
Published: December 2017

Vascular smooth muscle cells in atherosclerotic vessels proliferate and change from a contractile to a synthetic phenotype. To determine whether oxidized low-density lipoprotein (oxLDL) is involved in this transformation, we chronically incubated cultured smooth muscle cells with native and oxidized LDL. Western blot analysis detected a decrease in actin and myosin content in treated cells. This was dependent on the time and concentration of oxLDL employed. Confocal microscopic images of cells immunostained for smooth muscle-specific α-actin and myosin showed a normal, elongated alignment of myofilaments in cells after incubation with native LDL. Surprisingly, when the cells were treated with oxLDL, actin and myosin filaments underwent a striking process of disorganization and accumulation into ball-shaped aggregates. These changes were dependent on the duration and concentration of oxLDL employed. Our results demonstrate that oxLDL has the capacity to decrease the content of myofilaments in smooth muscle cells. The loss in myosin and actin protein may be associated with an unusual formation of large cellular aggregates that appear to be in the process of being expelled from the cell.
Translational mechanisms accelerate the rate of protein synthesis during canine pressure-overload hypertrophy
Authors: Nagatomo Yoshitatsu , Carabello Blase A. , Hamawaki Masayoshi , Nemoto Shintaro , Matsuo Takeshi , McDermott Paul J.
Published: December 2017

This study examined how translational mechanisms regulate the rate of cardiac protein synthesis during canine pressure overload in vivo. Acute aortic stenosis (AS) was produced by inflating a balloon catheter in the ascending aorta for 6 h; sustained AS was created by controlled banding of the ascending aorta. AS caused significant hypertrophy as reflected by increased left ventricular (LV) mass after 5 and 10 days. To monitor LV protein synthesis in vivo, myosin heavy chain (MHC) synthesis was measured by continuous infusion of radiolabeled leucine. Acute AS accelerated the rate of myosin synthesis without a corresponding increase in ribosomal RNA, indicating an increase in translational efficiency. Total MHC synthesis (mg MHC/LV per day) was significantly increased at 5 and 10 days of sustained AS. Total MHC degradation was not significantly altered at 5 days of AS but increased at 10 days of AS in concordance with a new steady state with respect to growth. Translational capacity (mg total RNA/LV) was significantly increased after 5 and 10 days of AS and was preceded by an increase in the rate of ribosome formation. MHC mRNA levels remained unchanged during AS. These findings demonstrate that cardiac protein synthesis is accelerated in response to pressure overload by an initial increase in translational efficiency, followed by an adaptive increase in translational capacity during sustained hypertrophic growth.
Role of reactive oxygen species in acetylcholine-induced preconditioning in cardiomyocytes
Authors: Yao Zhenhai , Tong Jiankun , Tan Xiaohui , Li Changqing , Shao Zuohui , Kim Woo Chan , vanden Hoek Terry L. , Becker Lance B. , Head C. Alvin , Schumacker Paul T.
Published: December 2017

We examined the ability of ACh to mimic ischemic preconditioning in cardiomyocytes and the role of ATP-sensitive potassium (KATP) channels and mitochondrial reactive oxygen species (ROS) in mediating this effect. Chick embryonic ventricular myocytes were studied in a flow-through chamber while flow rate, pH,[Formula: see text], and[Formula: see text] were controlled. Cell viability was quantified with propidium iodide (5 μM), and production of ROS was measured using 2′,7′-dichlorofluorescin diacetate. Data were expressed as means ± SE. Preconditioning with 10 min of ischemia followed by 10 min of reoxygenation or 10 min of ACh (1 mM) followed by a drug-free period before 1 h of ischemia and 3 h of reoxygenation reduced cell death to the same extent [preconditioning 19 ± 2% ( n = 6, P < 0.05) ACh 21 ± 5% ( n = 6, P < 0.05) vs controls 42 ± 5% ( n = 9)]. Like preconditioning, ACh increased ROS production threefold before ischemia [0.60 ± 0.16 ( n = 7, P< 0.05) vs. controls, 0.16 ± 0.03 ( n = 6); arbitrary units]. Protection and increased ROS production during ACh preconditioning were abolished with 5-hydroxydecanoate (5-HD, 100 μM), a selective mitochondrial KATPchannel antagonist, and the thiol reductant 2-mercaptopropionyl glycine (2-MPG, 1 mM), an antioxidant [cell death: 5-HD+ACh 37 ± 7% ( n = 5), 2-MPG+ACh 47 ± 6% ( n = 6); ROS signals: 5-HD+ACh 0.09 ± 0.03 ( n = 5), 2-MPG+ACh 0.01 ± 0.04 ( n = 4)]. In addition, ACh-induced ROS signaling was blocked by the mitochondrial site III electron transport inhibitor myxothiazol (0.02 ± 0.07, n = 5). These results demonstrate that activation of mitochondrial KATPchannels and increased ROS production from mitochondria are important intracellular signals that participate in ACh-induced preconditioning in cardiomyocytes.
Phosphatase inhibitor cantharidin blocks adenosine A1receptor anti-adrenergic effect in rat cardiac myocytes
Authors: Narayan Prakash , Mentzer Robert M. , Lasley Robert D.
Published: December 2017

Experiments were performed to examine whether the protein phosphatase inhibitor cantharidin blocks the anti-adrenergic effect of adenosine A1receptor stimulation. In electrically stimulated adult rat ventricular myocytes loaded with the intracellular calcium concentration ([Ca2+]i) indicator fluo-3, isoproterenol (10 nM) increased systolic [Ca2+]iby 46%, increased twitch amplitude by 56%, and increased total cellular cAMP content by 140%. The adenosine A1receptor agonist 2-chloro- N6-cyclopentlyadenosine (CCPA) reduced isoproterenol-stimulated [Ca2+]iand contractility by 87 and 80%, respectively, but reduced cAMP content by only 18%. Cantharidin had no effects on myocyte [Ca2+]i, contractility, or cAMP in the absence or presence of isoproterenol but blocked the effects of CCPA on [Ca2+]iand contractility by ∼44%. Cantharidin had no effect on CCPA attenuation of isoproterenol-induced increases in cAMP. Pretreatment with CCPA also reduced the increase in contractile parameters produced by the direct cAMP-dependent protein kinase A (PKA) activator 8-bromocAMP. These results suggest that activation of protein phosphatases mediate, in part, the anti-adrenergic effect of adenosine A1receptor activation in ventricular myocardium.
Arachidonic acid modulation of α1H, a cloned human T-type calcium channel
Authors: Zhang Yi , Cribbs Leanne L. , Satin Jonathan
Published: December 2017

Arachidonic acid (AA) and the products of its metabolism are central mediators of changes in cellular excitability. We show that the recently cloned and expressed T-type or low-voltage-activated Ca channel, α1H, is modulated by external AA. AA (10 μM) causes a slow, time-dependent attenuation of α1H current. At a holding potential of −80 mV, 10 μM AA reduces peak inward α1H current by 15% in 15 min and 70% in 30 min and shifts the steady-state inactivation curve −25 mV. AA inhibition was not affected by applying the cyclooxygenase inhibitor indomethacin or the lipoxygenase inhibitor nordihydroguaiaretic acid. The epoxygenase inhibitor octadecynoic acid partially antagonized AA attenuation of α1H. The epoxygenase metabolite epoxyeicosatrienoic acid (8,9-EET) mimicked the inhibitory effect of AA on α1H peak current. A protein kinase C (PKC)-specific inhibitor (peptide fragment 19–36) only partially antagonized the AA-induced reduction of peak α1H current and the shift of the steady-state inactivation curve but had no effect on 8,9-EET-induced attenuation of current. In contrast, PKA has no role in the modulation of α1H. These results suggest that AA attenuation and shift of α1H may be mediated directly by AA. The heterologous expression of T-type Ca channels allows us to study for the first time properties of this important class of ion channel in isolation. There is a significant overlap of the steady-state activation and inactivation curves, which implies a substantial window current. The selective shift of the steady-state inactivation curve by AA reduces peak Ca current and eliminates the window current. We conclude that AA may partly mediate physiological effects such as vasodilatation via the attenuation of T-type Ca channel current and the elimination of a T-type channel steady window current.
Altered frequency characteristic of central vasomotor control in SHR
Authors: Kuo Terry B. J. , Yang Cheryl C. H.
Published: December 2017

Previous work from our laboratory has demonstrated that the very low-frequency (VLF: 0–0.25 Hz) and low-frequency (LF: 0.25–0.8 Hz) power of arterial pressure variability (APV) are related to vasomotor reactivity in response to control signals from the rostral ventrolateral medulla (RVLM) via the sympathetic system in the rat. The present study evaluated the differences in the dynamic property of central vasomotor control between spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Experiments were carried out in 10- to 12-wk-old rats that were anesthetized with continuous infusion of pentobarbital sodium, paralyzed with pancuronium, and maintained on mechanical ventilation. We found that SHR exhibited significantly higher arterial pressure (AP), heart rate (HR), and VLF, LF, and high-frequency (0.8–2.4 Hz) power of APV than WKY under resting state. Broad-band electrical stimulation of the RVLM elicited parallel APV in the VLF and LF ranges in both rat strains. The evoked APV and transfer magnitude of the APV to stimulus spike rate variability (RVLM-AP magnitude) were significantly higher in SHR, especially in the LF range. The response frequency of central vasomotor control, represented by the high-cut frequency of RVLM-AP magnitude, was also extended in SHR. The disparity in RVLM-AP transfer magnitude between SHR and WKY became virtually absent after combined α- and β-adrenoceptor blockade by phentolamine and propranolol. These results suggest that the dynamic control of RVLM on AP reactivity is enhanced in SHR, in which the adrenergic system may play a major role.
Effect of acidosis on transient outward potassium current in isolated rat ventricular myocytes
Authors: Hulme J. T. , Orchard C. H.
Published: December 2017

The effect of acidosis on the transient outward K+ current ( Ito ) of rat ventricular myocytes has been investigated using the perforated patch-clamp technique. When the holding potential was −80 mV, depolarizing pulses to potentials positive to −20 mV activated Ito in subepicardial cells but activated little Ito in subendocardial cells. Exposure to an acid solution (pH 6.5) had no significant effect on Ito activated from this holding potential in either subepicardial or subendocardial cells. When the holding potential was −40 mV, acidosis significantly increased Ito at potentials positive to −20 mV in subepicardial cells but had little effect on Ito in subendocardial cells. The increase in Ito in subepicardial cells was inhibited by 10 mM 4-aminopyridine. In subepicardial cells, acidosis caused a +8.57-mV shift in the steady-state inactivation curve. It is concluded that in subepicardial rat ventricular myocytes acidosis increases the amplitude of Ito as a consequence of a depolarizing shift in the voltage dependence of inactivation.