bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of histidine 95 on pH gating of the cardiac gap junction protein connexin43.
Authors: Ek J F , Delmar M , Perzova R , Taffet S M
Published: June 2012

We have studied the role of histidine 95 (H95) on the pH gating of the cardiac gap junction protein connexin43 (Cx43). Wild-type and mutant rat cardiac Cx43 channels were expressed in antisense-injected Xenopus oocytes. Junctional conductance was measured using the dual voltage-clamp technique, and intracellular acidification was induced by superfusion with a sodium acetate-containing solution balanced at a pH of 6.2. H95 was substituted by other amino acids by use of oligonucleotide-directed site-specific mutagenesis. Replacing H95 for the hydrophobic residues methionine or phenylalanine, for the charged basic residue arginine, or for the noncharged residue glutamine (H95Q) yielded nonfunctional channels. Functional expression of H95Q was rescued by placing a histidine residue in position 93 (H95Q-L93H), 94 (H95Q-A94H), or 97 (H95Q-F97H) but not in position 96. Further experiments showed that replacing H95 with either aspartate (an acidic residue) or tyrosine (a polar uncharged residue) led to the expression of functional channels with a reduced susceptibility to acidification-induced uncoupling, whereas lysine (a basic residue) was more susceptible to uncoupling than the wild-type protein. The susceptibility to acidification-induced uncoupling was enhanced for the H95Q-A94H mutant when compared with the wild-type mutant, but it was significantly reduced when histidine was placed at position 93 (H95Q-L93H). Our data indicate that a properly placed histidine residue is an important structural element for functional expression as well as for pH regulation of Cx43. The results suggest that the importance of H95 on pH gating may be associated with a possible protonation of this residue on acidification of the intracellular environment.(ABSTRACT TRUNCATED AT 250 WORDS)
Intracellular oxidative stress induced by nitric oxide synthesis inhibition increases endothelial cell adhesion to neutrophils.
Authors: Niu X F , Smith C W , Kubes P
Published: June 2012

The objective of the present study was to determine whether prolonged inhibition of nitric oxide synthesis in endothelial cells increased the surface adhesion of these cells for neutrophils. Human umbilical vein endothelial cells (HUVECs) were grown to confluence in 48-well microtiter plates. Exposure of HUVECs to the nitric oxide synthesis inhibitor NG-nitro-L-arginine methyl ester (L-NAME) did not cause neutrophil adhesion at 1 hour but increased adhesion at 4 hours in a dose-dependent manner. The increased adhesion was prevented with L-arginine or nitric oxide donors but not an analogue of cGMP. The increased adhesion was inhibited by monoclonal antibodies directed against the beta 2-integrin CD18 and endothelial cell adhesion molecule ICAM-1. Platelet-activating factor (PAF) receptor antagonist WEB 2086 also prevented the L-NAME-induced neutrophil adhesion. Intracellular oxygen radical scavengers (dimethyl sulfoxide, butylated hydroxytoluene, and alpha, alpha'-dipyridyl), the iron chelator desferrioxamine, and the mitochondrial inhibitor azide inhibited the L-NAME-induced neutrophil adhesion, whereas extracellular oxygen radical scavengers (superoxide dismutase and catalase) had no effect. HUVECs were loaded with 2',7'-dichlorodihydrofluorescein diacetate, and oxidation to the fluorescent dichlorodihydrofluorescein (DCHF) was monitored. Fluorescence was enhanced in the L-NAME-treated HUVECs throughout the 4-hour incubation, an event inhibitable by an antioxidant and azide. The magnitude of the intracellular oxidation of DCHF was equivalent to approximately 0.8 mumol/L H2O2. These data suggest that prolonged nitric oxide synthesis inhibition in HUVECs causes an oxidant- and PAF-associated rise in adhesion on the surface of these endothelial cells for neutrophils.
Three-dimensional transmural mechanical interaction between the coronary vasculature and passive myocardium in the dog.
Authors: May-Newman K , Omens J H , Pavelec R S , McCulloch A D
Published: June 2012

The "garden hose" effect of coronary perfusion on diastolic left ventricular (LV) mechanics has been proposed to cause changes in systolic function by altering diastolic sarcomere length. We measured transmural distributions of three-dimensional shape change using radiopaque markers implanted in the LV free wall of eight isolated arrested canine hearts as functions of coronary arterial perfusion pressure (Pp) and LV pressure (PLV) and related these deformations to the local muscle fiber architecture. Increased Pp from 0 to 110 mm Hg produced a 10% reduction in LV chamber volume (P < .01) and 25% to 40% decreases in local three-dimensional wall strain at matched PLV, indicating myocardial stiffening. Significant decreases in the magnitudes of local deformation occurred preferentially in the cross-fiber and radial directions (P < .02), with no change in fiber strain. This suggests that changing coronary Pp does not alter diastolic fiber length; hence, the Frank-Starling law may not mediate the Gregg effect. Since the myocardial microvessels are primarily oriented parallel to the muscle fibers, the observed myocardial stiffening occurs in the directions transverse to the microvessels rather than along their length. Local myocardial wall volume in the unloaded LV demonstrated a uniform 5% increase from the unperfused state to Pp of 50 mm Hg. With further increases in Pp up to 110 mm Hg, the change in regional wall volume from the unperfused state developed a substantial transmural gradient increasing by 7% at the epicardium and 15% at the subendocardium. This reflects a significant increase (P < .02) in intramyocardial coronary capacitance from epicardium to endocardium, which may be related to a transmural gradient in coronary distensibility or vascularity.
Responses of neurons in the nucleus tractus solitarius to stimulation of heart and lung receptors in the rat.
Authors: Hines T , Toney G M , Mifflin S W
Published: June 2012

To characterize central integration of reflex responses to stimulation of mechanically and chemically sensitive receptors in the heart and lung, male rats (350 to 425 g) were anesthetized (pentobarbital, 50 mg/kg IP) and paralyzed (gallamine triethiodide, 25 mg/kg IV) and then they underwent bilateral sinoaortic denervation. Extracellular activity of neurons in the nucleus tractus solitarius (NTS) was recorded in response to bolus intra-atrial saline (50, 100, 200, or 300 microL) or phenylbiguanide (PBG, 16 micrograms/kg in 100 microL) administered in random sequence. Changes in mean arterial pressure (MAP), mean right atrial pressure, and right atrial systolic pressure (RASP) were measured as correlates of stimulus intensity, and heart rate (HR) and renal sympathetic nerve activity (RSNA) were used to assess efferent reflex effects of cardiac and pulmonary receptor stimulation. NTS neurons with possible afferent input from stretch and chemically sensitive receptors were identified by an excitatory evoked response to electrical stimulation of the ipsilateral vagus nerve (1 Hz, 500 microA, 1-millisecond duration). Thirty-eight vagus nerve-evoked NTS units with onset latencies of 25.3 +/- 0.9 milliseconds displayed excitatory or inhibitory responses to saline or PBG injections or to both interventions. Saline administration elicited volume-dependent transient increases in MAP and RASP, which were followed by reflex decreases in MAP, HR, and RSNA. PBG injections also evoked hypotension, bradycardia, and sympathoinhibition.(ABSTRACT TRUNCATED AT 250 WORDS)
Effects of III-IV linker mutations on human heart Na+ channel inactivation gating.
Authors: Hartmann H A , Tiedeman A A , Chen S F , Brown A M , Kirsch G E
Published: June 2012

Na+ channel inactivation, a critical determinant of refractoriness, differs in cardiomyocytes and neurons. In rat brain type IIa (rB2a) Na+ channels, a critical residue in the cytoplasmic linker between domains III and IV regulates fast inactivation such that a Phe-->Gln substitution (F1489Q) inhibits inactivation by at least 85%. Since this residue is conserved in voltage-gated Na+ channels, we tested whether F1485Q, the analogous mutation in human heart (hH1a) Na+ channels, has a similar functional effect. We found that fast inactivation in wild-type (WT) channels expressed in Xenopus oocytes was complete within 15 milliseconds at a test potential of 0 mV, and its time course was biexponential with time constants of 0.4 and 2 milliseconds. But in contrast to rB2a, the FQ mutation inhibited inactivation by < 50% and increased mean single-channel open time by only twofold. Residual fast inactivation was monoexponential, with a time constant similar to that of the slower phase of normal inactivation (2 milliseconds). In the mutant channels, unlike WT, null tracings were absent at holding potentials in the range of -140 to -120 mV, and the voltage range of steady-state inactivation coincided exactly with that of activation, suggesting that residual inactivation was tightly coupled to the open state. As in rB2a, simultaneous mutations of I1484Q and M1486Q, in addition to mutation F1485Q, completely inhibited fast inactivation. Our results show that in heart Na+ channels, the IFM cluster controls the stability of both open- and closed-channel inactivation in a manner qualitatively similar to that in the brain. Structural differences in the putative inactivation receptor may explain the distinct gating patterns in channel subtypes.
Platelet tyrosine kinases and fibrinogen receptor activation.
Authors: Furman M I , Grigoryev D , Bray P F , Dise K R , Goldschmidt-Clermont P J
Published: June 2012

Platelet adhesion and aggregation during hemostasis and thrombosis are usually limited to sites where the integrity of the vessel wall is disrupted. The high concentration of platelet agonists within these sites represents a putative control mechanism for targeting platelet activation. Although much has been learned about the intracellular signaling systems controlling platelet activation, our understanding of the connection between signaling molecules and platelet aggregation remains limited. Tyrosine kinases are important signaling enzymes in cells and are abundant in platelets. Previous reports indicate that binding of glycoprotein IIb-IIIa (GPIIb-IIIa) to fibrinogen can induce the tyrosine phosphorylation of specific substrates. We show that, in turn, protein tyrosine kinase activity is necessary for agonist-induced activation of GPIIb-IIIa. Genistein and the tyrphostin AG-18 are two specific tyrosine kinase inhibitors, and the former has been shown to inhibit platelet aggregation. We use genistein and AG-18 in the present study to demonstrate that aggregation inhibition is due to suppression of GPIIb-IIIa activation. In contrast, genistin, an isoflavone compound related to genistein, and acetylsalicylic acid do not affect the tyrosine kinase-signaling pathway, nor do they inhibit GPIIb-IIIa activation induced by strong agonists. On identifying prominent tyrosine kinase substrates in activated platelets, we confirm that several substrates correspond to proteins associated with the cytoskeleton: the 85-kD subunit of phosphatidylinositol 3-kinase, the SH3-containing and actin-associating p85, pp60Src, and pp125FAK.(ABSTRACT TRUNCATED AT 250 WORDS)
Quantitative analysis of potassium channel mRNA expression in atrial and ventricular muscle of rats.
Authors: Dixon J E , McKinnon D
Published: June 2012

The expression of 15 different potassium channel genes in rat atrial and ventricular muscle was quantitatively compared by use of an RNase protection assay. Of these genes, only five, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2, were expressed at significant levels in cardiac muscle. In comparisons of atrial and ventricular RNA samples, transcripts from the Kv1.2 and Kv4.2 genes showed the largest differences in relative abundance. There was an approximately twofold decrease in total Kv4 subfamily mRNA expression in atrial muscle relative to ventricular muscle and a 70% increase in total Kv1 subfamily mRNA. Variation of potassium channel mRNA expression within the left ventricular wall was also examined. There was a large gradient of Kv4.2 expression across the ventricular wall, and Kv4.2 expression in epicardial muscle was more than eight times higher than in papillary muscle. Other potassium channel genes were expressed at relatively uniform levels across the ventricular wall. The results suggest that transcriptional regulation makes a significant contribution to the control of potassium channel expression in cardiac muscle and to the variation of the electrophysiological phenotype of myocytes from different regions of the myocardium.
Hypoxia induces apoptosis with enhanced expression of Fas antigen messenger RNA in cultured neonatal rat cardiomyocytes.
Authors: Tanaka M , Ito H , Adachi S , Akimoto H , Nishikawa T , Kasajima T , Marumo F , Hiroe M
Published: June 2012

We examined whether apoptosis occurs in cardiomyocytes by hypoxia in vitro. Neonatal rat cardiomyocytes and nonmyocytes were cultured in 95% N2-5% CO2 atmosphere to produce hypoxic conditions. DNA fragmentation into integer multiples of the internucleosomal DNA length was observed in cardiomyocytes as early as 12 hours, whereas nonmyocytes did not show fragmentation of DNA up to 72 hours. DNA fragmentation of cardiomyocytes induced by hypoxia was also confirmed by nick-end labeling in situ. Messenger RNA for Fas antigen, a mediator of apoptotic cell death, was expressed in both cardiomyocytes and nonmyocytes as revealed by Northern blotting and in situ hybridization. In hypoxic condition, Fas messenger RNA levels in cardiomyocytes were upregulated by twofold over controls, whereas those of nonmyocytes were downregulated. These results indicate that cardiomyocyte death by hypoxia can occur via apoptosis and that Fas antigen may be associated with the mechanism of this apoptotic process.
Reduced unloaded sarcomere shortening velocity and a shift to a slower myosin isoform in acute murine coxsackievirus myocarditis.
Authors: Hamrell B B , Huber S A , Leslie K O
Published: June 2012

We developed a mouse myocardial preparation to study cellular dysfunction in acute coxsackievirus myocarditis. Thin right ventricular papillary muscles from normal mice (n = 8) were compared with muscles from mice 7 days after coxsackievirus infection (n = 7). Sarcomere shortening was studied with laser diffraction (HeNe, lambda = 623.8 nm). A servomotor was used to shorten a muscle until slack early in isometric contraction. Unloaded sarcomere shortening velocity (Vo) was measured at the start of zero force at slack length. Vo was independent of the extent of slack release and was the same as that estimated with an isotonic force-sarcomere shortening velocity relation. Resting muscle stiffness was calculated from shortening perturbations in resting muscles. The histology of some papillary muscles (normal, n = 4; infected, n = 3) was studied. There was no ventricular hypertrophy. Resting sarcomere length (SL) in infected preparations (2.11 +/- 0.07 micron) (mean +/- 1 SD) was the same as in normal preparations (2.11 +/- 0.08 micron). In isometric twitches in normal and infected muscles, total peak force (4.31 +/- 1.07 and 3.77 +/- 1.86 g/mm2, respectively) resting force (0.81 +/- 0.37 and 0.81 +/- 0.35 g/mm2, respectively), and time to peak force (129.5 +/- 20.3 and 125.2 +/- 13.0 milliseconds, respectively) were not significantly different. Vo was 4.14 +/- 0.84 micron/s in normal muscles at an SL of 2.08 +/- 0.09 micron and 1.70 +/- 0.33 micron/s in infected muscles at an SL of 2.06 +/- 0.08 micron. Resting stiffness was the same for normal and infected muscles. There was inflammation but no fibrosis or necrosis. Thus, Vo was depressed early in acute viral myocarditis without hypertrophy, myocyte necrosis, fibrosis, or altered resting stiffness. Pyrophosphate gel electrophoresis showed a shift from predominantly fast to slow myosin isoforms. Apparently, there is remodeling of the contractile apparatus early in acute coxsackievirus myocarditis that is caused either by the direct effects of the virus or the immune response.
Pertussis toxin-sensitive G protein mediates coronary microvascular control during autoregulation and ischemia in canine heart.
Authors: Komaru T , Wang Y , Akai K , Sato K , Sekiguchi N , Sugimura A , Kumagai T , Kanatsuka H , Shirato K
Published: June 2012

GTP-binding regulatory proteins (G proteins) regulate various biological functions, but their participation in controlling coronary microvascular tone has not been established yet. The goal of the present study was to elucidate the role of pertussis toxin (PTX)-sensitive G protein in regulating coronary microvascular tone during autoregulation and ischemia. In 42 open-chest dogs, coronary arterial microvessels on the surface of the left ventricle were directly observed by epi-illuminated fluorescence microangiography using a floating objective system. PTX (300 ng/mL) was superfused onto the surface of the left ventricle for 2 hours to block Gi and G(o) protein in epimyocardial coronary microvessels in vivo. PTX superfusion caused no change in the resting diameters of microvessels and significantly blocked the vasoconstriction induced by BHT 920 (a selective alpha 2-agonist). After pretreatment with PTX or its vehicle, the left anterior descending coronary artery (LAD) was occluded by a hydraulic occluder to reduce coronary perfusion pressure (CPP) in a stepwise fashion. A mild stenosis (CPP, 60 mm Hg), a severe stenosis (CPP, 40 mm Hg), and complete occlusion were sequentially produced. Coronary flow velocity in the LAD distal to the stenotic site was continuously monitored. In both PTX and vehicle groups, flow velocity did not significantly decrease during mild stenosis, proving that transmural coronary autoregulatory function was well preserved in the preparation. During severe stenosis and complete occlusion, the coronary flow velocity significantly decreased. In the vehicle group, microvessels < 100 microns in inner diameter significantly dilated in response to the reduction in perfusion pressure (mild stenosis, 6.2 +/- 1.9%; severe stenosis, 21.1 +/- 4.4%; and complete occlusion, 16.8 +/- 5.9%; P < .05 versus baseline diameters). In the PTX group, microvessels did not dilate during each occlusion level (mild stenosis, -2.0 +/- 0.9%; severe stenosis, -3.9 +/- 1.9%; and complete occlusion, -13.4 +/- 2.9%; P < .05 versus vehicle group). PTX did not affect the microvascular dilation caused by nitroprusside. The present data indicate that PTX-sensitive G protein is crucially involved in microvascular control during autoregulation and ischemia.
Molecular cloning, expression, and chromosomal localization of two isoforms of the AE3 anion exchanger from human heart.
Authors: Yannoukakos D , Stuart-Tilley A , Fernandez H A , Fey P , Duyk G , Alper S L
Published: June 2012

Cl-/HCO3- exchange contributes to regulation of pHi and [Cl-] in cardiac muscle, with possible effects on excitability and contractility. We have isolated human heart cDNAs, which encode two isoforms of the anion exchanger AE3. These clones share long portions of common sequence but have different 5' ends encoding distinct amino-terminal amino acid sequences. The longer AE3 polypeptide of 1232 amino acids, bAE3, displays nearly 96% amino acid sequence identity to the rat and mouse AE3 "brain isoforms." The shorter cAE3 polypeptide of 1034 amino acids in length corresponds to the rat AE3 "cardiac isoform." The unique N-terminal 73 amino acids of the cAE3 sequence are less well conserved between rat and human. Northern blot analysis with isoform-specific probes revealed the presence of both cAE3 and bAE3 mRNAs in human heart tissue. Both AE3 protein isoforms were overexpressed in Chinese hamster ovary cells and detected by immunoblot with antipeptide antibodies. Immunoblot studies of human cardiac membranes detected only cAE3 polypeptides, which were apparently not susceptible to enzymatic deglycosylation. Injection into Xenopus oocytes of cRNAs encoding either cAE3 or bAE3 produced increased 36Cl- uptake into the oocytes, confirming the ability of both AE3 isoforms to transport Cl-. The human AE3 gene was localized to chromosome 2. AE3 may provide a new pharmacologic target for antiarrhythmic and cardioprotective drugs.
Effects of heparin and N-acetyl heparin on ischemia/reperfusion-induced alterations in myocardial function in the rabbit isolated heart.
Authors: Friedrichs G S , Kilgore K S , Manley P J , Gralinski M R , Lucchesi B R
Published: June 2012

Evidence is presented that heparin pretreatment produces protective effects on myocardial tissue distinct from its anticoagulant activity. The present study examines the ability of heparin sulfate and N-acetyl heparin (a derivative of heparin devoid of anticoagulant effects) to protect the heart from injury associated with global ischemia and reperfusion. Male New Zealand White rabbits were administered either heparin sulfate (n = 7, 300 U/kg i.v.), N-acetyl heparin (n = 6, 1.73 mg/kg i.v.), or vehicle (n = 6). Two hours after treatment, the hearts were removed, perfused on a Langendorff apparatus, and subjected to 30 minutes of global ischemia, followed by 45 minutes of reperfusion. During reperfusion, creatine kinase concentrations in the coronary sinus effluent were greater in hearts from vehicle-treated rabbits compared with hearts from N-acetyl heparin-treated and heparin-treated rabbits. Left ventricular end-diastolic pressure after 45 minutes of reperfusion in the vehicle-treated group was 64 +/- 15 mm Hg compared with 17 +/- 4 and 10 +/- 3 mm Hg in the heparin-pretreated and N-acetyl heparin-pretreated groups, respectively. Heparin, but not N-acetyl heparin, increased the activated partial thromboplastin time, consistent with its known anticoagulant action. Heparin and N-acetyl heparin inhibited complement-mediated erythrocyte lysis in a concentration-dependent manner. The glycosaminoglycans, in contrast to r-hirudin, reduced complement activation-induced injury in the rabbit isolated heart. The results demonstrate that heparin or N-acetyl heparin, administered to the intact rabbit, protects the isolated heart from subsequent myocardial dysfunction secondary to ischemia/reperfusion. The cardioprotective effects of heparin and N-acetyl heparin are independent of an antithrombin mechanism.
Calcium-activated chloride current in rabbit coronary artery myocytes.
Authors: Lamb F S , Volk K A , Shibata E F
Published: June 2012

Whole-cell patch-clamp techniques were used to study enzymatically dispersed epicardial coronary artery smooth muscle cells. Depolarizing voltage pulses of 500-millisecond duration from -60 mV (118 mmol/L CsCl, 22 mmol/L tetraethylammonium chloride, and 5 mmol/L EGTA pipette solution) elicited inward L-type calcium currents (ICa). When EGTA was omitted from the pipette solution, an outward current was superimposed on the calcium current, and repolarizing voltage steps produced an inward tail current (IT). The amplitude of these inward currents was proportional to the ICa amplitude from -30 to +50 mV. The time course of decay of the current was well fit by a single exponential equation. The time constant (tau) of this equation did not change with the size of IT but was clearly voltage dependent (shorter at more negative potentials). Changing the chloride reversal potential from -1.3 to -39.7 mV by anion substitution using methanesulfonate as the chloride replacement in the pipette solution shifted the zero current level of IT from 0.9 +/- 0.56 to -33.1 +/- 0.85 mV. The tail current was blocked by nifedipine (10(-6) mol/L) and by isosmolar calcium substitution with barium in the bath solution and was enhanced by the dihydropyridine agonist Bay K 8644 (10(-6) mol/L). IT was also blocked by the chloride channel blockers DIDS (10(-4) mol/L) and niflumic acid (10(-5) mol/L). Caffeine (10(-2) mol/L), which releases intracellular calcium stores, caused an inward current at holding potentials (-60 mV), which was inhibited by DIDS. Caffeine also inhibited subsequent attempts to elicit IT by depolarizing pulses (88% reduction in IT).(ABSTRACT TRUNCATED AT 250 WORDS)
Multiple factors contribute to acetylcholine-induced renal afferent arteriolar vasodilation during myogenic and norepinephrine- and KCl-induced vasoconstriction. Studies in the isolated perfused hydronephrotic kidney.
Authors: Hayashi K , Loutzenhiser R , Epstein M , Suzuki H , Saruta T
Published: June 2012

Acetylcholine (ACh) elicits vasodilation by releasing a number of endothelium-derived relaxing factors (EDRFs). We used the isolated perfused hydronephrotic rat kidney to examine the characteristics of ACh-induced vasodilation of renal afferent arterioles during different types of underlying vasoconstriction. Basal arteriolar tone was increased by either elevating perfusion pressure to 180 mm Hg (myogenic), administering 0.3 mumol/L norepinephrine (NE), or elevating medium potassium concentration to 30 mmol/L (KCl). ACh (10 mumol/L) completely reversed myogenic and NE-induced vasoconstriction and reversed KCl-induced vasoconstriction by 80 +/- 5%. However, whereas ACh produced a sustained vasodilation during KCl- and NE-induced vasoconstriction, only a transient reversal of myogenic vasoconstriction was observed, and myogenic tone recovered within 5 to 10 minutes. ACh-induced vasodilation of arterioles preconstricted with KCl was markedly inhibited by either indomethacin (100 mumol/L) or nitro-L-arginine (100 mumol/L) and was completely abolished by pretreatment with both inhibitors. In contrast, indomethacin and nitro-L-arginine had no effect on the transient response to ACh observed during pressure-induced vasoconstriction. In vessels preconstricted with NE, nitro-L-arginine converted the normally sustained response to ACh to a transient vasodilation, which was refractory to both nitric oxide synthase and cyclooxygenase inhibition. Since this component was not observed during KCl-induced vasoconstriction, it may reflect the actions of an, as yet unidentified, endothelium-derived hyperpolarizing factor (EDHF). Our findings thus suggest that prostanoids, nitric oxide, and EDHF all contribute to ACh-induced renal afferent arteriolar vasodilation and that the relative contributions of these individual EDRFs depends on the nature of the underlying renal vascular tone.
Augmented receptor-mediated Ca2+ mobilization causes supersensitivity of contractile response to serotonin in atherosclerotic arteries.
Authors: Miwa Y , Hirata K , Matsuda Y , Suematsu M , Kawashima S , Yokoyama M
Published: June 2012

We have previously reported that atherosclerotic arteries obtained from Watanabe heritable hyperlipidemic (WHHL) rabbits exhibit a marked increase of contractile response to serotonin (5-hydroxytryptamine [5-HT]) and ergonovine and that these augmented contractile responses to specific agonists may play an important role in the pathogenesis of vasospasm. In the present study, we investigated whether supersensitivity to 5-HT in atherlosclerotic arteries was due to an increase in 5-HT receptor-mediated Ca2+ mobilization or to an increase in Ca2+ sensitivity of the contractile elements. We measured simultaneously both isometric tension and [Ca2+]i in fura 2-loaded aortic smooth muscle strips from control and WHHL rabbits. Muscle tension in the high K+ (72.7 mmol/L)-stimulated states and [Ca2+]i in both resting and high K(+)-stimulated states did not differ between control and WHHL rabbits. In atherosclerotic aortas from WHHL rabbits, the dose-response curves of both tension and [Ca2+]i for 5-HT were shifted to the left at lower threshold concentrations and one-half maximally effective dose. The maximum response of contraction produced by 5-HT in WHHL rabbits was augmented compared with that in control rabbits (123 +/- 17% versus 33 +/- 7% of the 72.7 mmol/L K(+)-induced contraction, P < .001). The maximum response of [Ca2+]i produced by 5-HT was also augmented in WHHL rabbits compared with control rabbits (29 +/- 4% versus 10 +/- 0.9% of the 72.7 mmol/L K(+)-induced [Ca2+]i, P < .001). In contrast, the responses of contraction and [Ca2+]i to phenylephrine were similar between control and WHHL rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)
Regulation of smooth muscle alpha-actin promoter by vasopressin and platelet-derived growth factor in rat aortic vascular smooth muscle cells.
Authors: Van Putten V , Li X , Maselli J , Nemenoff R A
Published: June 2012

Vasoconstrictors such as arginine vasopressin (AVP) and angiotensin II (Ang II) have been shown to increase protein and mRNA levels of smooth muscle alpha-actin (SM-alpha-actin) in vascular smooth muscle cells. In the same cells, platelet-derived growth factor (PDGF) decreased SM-alpha-actin protein and mRNA. The rat SM-alpha-actin promoter that has recently been isolated contains two E-boxes and three CC(A/T)6GG (CArG) elements. To examine regulation of the SM-alpha-actin promoter, a 765-bp region of the rat SM-alpha-actin gene was ligated into chloramphenicol acetyltransferase (CAT)-containing vectors and transfected into rat aortic vascular smooth muscle cells. Stimulation of cells with either AVP or Ang II increased CAT activity 5- to 10-fold. PDGF was able to completely block the AVP-induced increase in CAT activity. To identify regions of the promoter responsible for both the AVP stimulation and PDGF inhibition of promoter activity, a series of truncation mutants were prepared and transfected into vascular smooth muscle cells. Truncation of both E-boxes and the most distal CArG element did not qualitatively alter either AVP-induced stimulation of CAT activity or PDGF inhibition. However, removal of the middle CArG element resulted in a loss of AVP stimulation. These studies indicate that the AVP-induced elevation and PDGF-induced inhibition of SM-alpha-actin levels in vascular smooth muscle cells are mediated at least in part through regulation of the SM-alpha-actin promoter. The critical region of the promoter mediating this effect involves at a minimum one of the CArG elements.
Effects of pHi on isometric force and [Ca2+]i in porcine coronary artery smooth muscle.
Authors: Nagesetty R , Paul R J
Published: June 2012

During ischemia or hypoxia, alterations in pHi may play a significant role in alteration of vessel wall function. We studied the effects of altering pHi on isometric force and [Ca2+]i in porcine coronary artery. pHi was altered at constant pHo by use of NH4Cl and measured with the fluorescent dye BCECF. [Ca2+]i was monitored with fura 2 and ratiometric fluorescence measurements. Addition of NH4Cl elicited a concentration-dependent (2 to 30 mmol/L) sustained increase in isometric force in unstimulated tissues. In tissues stimulated with KCl (29 mmol/L) or U46619 (1 mumol/L), addition of NH4Cl elicited a rapid but transient decrease followed by a sustained increase in force above the initial stimulated levels. Removal of NH4Cl was associated with a transient decrease and increase followed by a prolonged depression of force and slow recovery to initial levels. Addition of NH4Cl elicited a rapid monotonic increase in pHi and then a slow recovery toward initial levels; washout of NH4Cl led to a rapid acidification followed by recovery. In contrast to the steady state effects of NH4Cl on force, its effects on [Ca2+i were in the opposite direction. During the sustained increase in force elicited by NH4Cl alkalinization, [Ca2+]i was substantially decreased, whereas when force was depressed during the acidification elicited by NH4Cl washout, [Ca2+i was increased to values observed before addition of NH4Cl. The initial transients in force elicited by NH4Cl addition or washout were also associated with opposite changes in [Ca2+]i. Thus, the effects on force of the NH4Cl-induced changes in pHi are associated with changes in the Ca2+ sensitivity of the contractile apparatus rather than mediated through changes in [Ca2+]i.
K
+
Channel Blockers Inhibit Tissue Factor Expression by Human Monocytic Cells
Authors: Crutchley David J. , Conanan Lobella B. , Que Benito G.
Published: June 2012

Abstract
Human monocytes express the important procoagulant protein, tissue factor (TF), after stimulation by a variety of agents, including bacterial lipopolysaccharide (LPS). Monocyte TF expression may contribute to intravascular coagulation in a number of disease states. The present studies show that monocytic cell TF expression can be inhibited by several agents known to block cellular K
+
channels. Exposure of human peripheral blood to 100 ng/mL LPS for 2 hours led to pronounced TF procoagulant activity associated with the mononuclear cell fraction. This was inhibited by 4-aminopyridine (2 mmol/L), tetraethylammonium chloride (10 mmol/L), and apamin (1 μmol/L). In contrast, charybdotoxin (100 nmol/L) was inactive. More detailed studies were carried out in cultured human monocytic tumor THP-1 cells. These cells exhibited low but detectable levels of TF mRNA, measured by reverse transcription and polymerase chain reaction; cell surface procoagulant activity, measured by a plasma clotting assay; and cell homogenate TF antigen, measured by immunoassay. Exposure of THP-1 cells to 1 μg/mL LPS led to threefold to fivefold increases in all three parameters. Basal and LPS-induced levels of all three parameters were reduced in a dose-dependent manner by 4-aminopyridine (I
50
, 1 mmol/L) and tetraethylammonium chloride (I
50
, 20 mmol/L) but not by apamin or charybdotoxin. Expression of TF activity was also inhibited by glibenclamide, an inhibitor of ATP-dependent K
+
channels (I
50
, 25 μmol/L). These results suggest that facilitation of TF synthesis may be an important role for K
+
channels in monocytes.
Coexpression of P
2Y
and P
2U
Receptors on Aortic Endothelial Cells
Authors: Communi Didier , Raspe Eric , Pirotton Sabine , Boeynaems Jean-Marie
Published: June 2012

Abstract
Depending on the vascular bed considered, the actions of ATP on the endothelium are mediated by either P
2Y
or P
2U
receptors. The two types of receptors seem to coexist on bovine aortic endothelial cells, where they are both coupled to phospholipase C. In this study, we have investigated whether they are truly coexpressed on the same cells and whether their signaling pathways diverge beyond phospholipase C activation. Measurements of [Ca
2+
]
i
in single cells showed that almost all bovine aortic endothelial cells are responsive to both 2-methylthio-ATP (2MeSATP), an agonist of P
2Y
receptors, and UTP, an agonist of P
2U
receptors. UTP stimulated the release of prostacyclin from freshly isolated bovine aortic endothelial cells, even when they were exposed to cycloheximide at the time of their collection: this indicates that P
2U
receptors must already be expressed on endothelial cells in situ and do not appear during cell culture. The time course of inositol phosphate (InsP) accumulation and the relative proportion of Ins(1,4,5)P
3
, Ins(1,3,4,5)P
4
, and Ins(1,3,4)P
3
were similar in cells stimulated by 2MeSATP or UTP. UTP and 2MeSATP both stimulated the hydrolysis of phosphatidylcholine by phospholipase D, as reflected by the release of [
3
H]choline from prelabeled cells. The responses to both agents were blocked after downregulation of protein kinase C, resulting from a prolonged exposure to phorbol 12-myristate 13-acetate: this blockade occurred at a step distal to phospholipase C activation. A single difference between the two pathways has been identified: the effect of 2MeSATP on InsP
3
was significantly more inhibited after a short exposure to phorbol 12-myristate 13-acetate than that of UTP. This discrepancy is consistent with the involvement of distinct G proteins in the activation of phospholipase C by P
2Y
and P
2U
receptors.
Nitric Oxide–Generating Compounds Inhibit Total Protein and Collagen Synthesis in Cultured Vascular Smooth Muscle Cells
Authors: Kolpakov Valeri , Gordon David , Kulik Thomas J.
Published: June 2012

Abstract
Nitric oxide (NO) participates in the regulation of vascular tone and smooth muscle cell proliferation, but little is known of its effect on total protein and matrix synthesis in smooth muscle. We studied the effects of the NO-generating compounds
S
-nitroso-
N
-acetylpenicillamine (SNAP, 0.4 to 1.2 mmol/L) and sodium nitroprusside (SNP, 0.1 to 0.5 mmol/L) on total protein (using [
3
H]leucine) and collagen (using [
3
H]proline) synthesis in cultured rabbit aortic smooth muscle cells. Both agents caused dose-dependent inhibition of the relative rate of protein (maximum reduction of 87% [SNAP] and 80% [SNP]) and collagen synthesis, as measured by trichloroacetic acid–precipitated label. The magnitudes of percent inhibition of total protein and collagen synthesis were approximately equal. Inhibition of protein synthesis by SNAP and SNP was prevented by hemoglobin (10 μmol/L), suggesting that the protein synthesis inhibition was due to NO release. Inhibition of protein synthesis was reversible after removal of SNAP and SNP and was not caused by damage to the cells. These results suggest that NO may function as a modulator of vascular smooth muscle cell protein synthesis and production of extracellular matrix components.
Stable Expression and Coupling of Cardiac L-Type Ca
2+
Channels With β
1
-Adrenoceptors
Authors: Yatani Atsuko , Wakamori Minoru , Niidome Tetsuhiro , Yamamoto Satoshi , Tanaka Isao , Mori Yasuo , Katayama Kouich , Green Stuart
Published: June 2012

Abstract
A number of neurotransmitters modulate cardiac dihydropyridine-sensitive L-type Ca
2+
channels through several homologous G protein–coupled receptors. Previous studies that have examined receptor–Ca
2+
channel interactions have suffered because of the coexpression of various receptor subtypes in native cells. To study the functional coupling of a particular receptor subtype to these channels, rabbit cardiac Ca
2+
channel α
1
and skeletal β and α
2
/δ subunits were stably expressed in baby hamster kidney cells. In this stable cell line, Ca
2+
channels remained at high levels (>1000 fmol/mg protein, or 2700 channels per cell) over extended times. The expressed recombinant Ca
2+
channels displayed the voltage dependence of activation and inactivation, unitary conductance, and pharmacology characteristic of native cardiac L-type Ca
2+
channels. Subsequent coexpression of the β
1
-adrenoceptors (150 to 300 fmol/mg protein) with the Ca
2+
channels resulted in cell responsiveness to the extracellular application of isoproterenol. These results indicate that heterogeneous expression in mammalian cells provides a useful system for studying both biophysical analysis of Ca
2+
channel properties and receptor-coupled regulatory processes.
Inhibition of Nitric Oxide Synthesis Causes Myocardial Ischemia in Endotoxemic Rats
Authors: Avontuur J. A. M. , Bruining H. A. , Ince C.
Published: June 2012

Abstract
Inhibitors of nitric oxide (NO) synthesis have been used in the treatment of septic and endotoxic shock. However, several studies question the beneficial effect of inhibiting NO production in sepsis and endotoxemia. We have investigated the effect of inhibition of NO synthesis after endotoxemia in the isolated perfused rat heart. In hearts from endotoxin-treated animals, coronary flow was elevated 64% and oxygen consumption was elevated 20% compared with control hearts. NADH fluorescence imaging was used as an indicator of regional hypoperfusion. A homogeneous low-surface NADH fluorescence, indicative of adequate tissue perfusion, was observed in both control and endotoxin-treated hearts. The increase in coronary flow and oxygen consumption could only partially be prevented by pretreatment of the animals with dexamethasone. Addition of
N
ω
-nitro-
l
-arginine (NNLA), an inhibitor of NO synthesis, to the perfusion medium eliminated differences in coronary flow and oxygen consumption between normal and endotoxin-treated hearts. However, NADH surface fluorescence images of endotoxin-treated hearts after NNLA revealed areas of high fluorescence, indicating local ischemia, whereas the control hearts remained without signs of ischemia. The ischemic areas were present at various perfusion pressures and disappeared after the infusion of
l
-arginine, the natural precursor of NO, or the exogenous NO donor sodium nitroprusside. Methylene blue (MB), an inhibitor of soluble guanylate cyclase, the effector enzyme of NO, also eliminated differences in coronary flow and produced similar areas of local myocardial ischemia in endotoxin-treated hearts but not in control hearts. Reducing coronary flow by direct vasoconstriction with vasopressin resulted in similar patterns of myocardial ischemia as with NNLA and MB. Our results suggest that the coronary vasodilation in the isolated rat heart after endotoxemia is caused by an increased release of NO. Coronary flow reduction with NNLA, MB, or direct vasoconstriction with vasopressin causes local areas of myocardial ischemia in endotoxin-treated hearts but not in untreated hearts. These data suggest that endotoxemia promotes myocardial ischemia in vulnerable areas of the heart after inhibition of the NO pathway or direct vasoconstriction.
Modulation of Baroreceptor Activity by Nitric Oxide and
S
-Nitrosocysteine
Authors: Matsuda Tadashi , Bates James N. , Lewis Stephen J. , Abboud Francois M. , Chapleau Mark W.
Published: June 2012

Abstract
The goal of this study was to determine whether nitric oxide (NO) and the NO donor,
S
-nitrosocysteine (cysNO), modulate the activity of carotid sinus baroreceptors. Baroreceptor activity was recorded from the vascularly isolated carotid sinus in anesthetized rabbits. Baroreceptor activity decreased in a dose-dependent manner after injection of either NO or cysNO as constant pressure was maintained, and activity recovered spontaneously over time, within seconds to minutes. The baroreceptor pressure-activity relation was shifted significantly to the right by cysNO, with a profound suppression of activity at high pressure. Baroreceptor activity at 160 mm Hg averaged 76±8%, 60±6%, and 36±5% of the control maximum during exposure to 10
−4
, 2 to 3×10
−4
, and 10
−3
mol/L cysNO, respectively. The inhibition of activity by the
l
and
d
isomers of cysNO was equivalent and was blocked by reduced hemoglobin, suggesting that the effect was mediated by NO. The suppression of baroreceptor activity by cysNO was not related to vascular relaxation as measured by videomicrometer. Inhibition of soluble guanylate cyclase with methylene blue or 6-anilinoquinoline-5,8-quinone (LY83583, 10
−5
mol/L) did not attenuate and dibutyryl cGMP (10
−3
mol/L) did not mimic the suppression of baroreceptor activity by cysNO, suggesting a cGMP-independent mechanism. Activation of endogenous NO formation with thimerosal (10
−5
to 10
−4
mol/L) reduced maximum baroreceptor activity in five of eight experiments to 59±7% of the control maximum. The NO synthase inhibitor nitro-
l
-arginine methyl ester (L-NAME, 10
−4
mol/L) by itself failed to influence baroreceptor activity but prevented thimerosal-induced suppression of activity. Addition of
l
-arginine (10
−3
mol/L) after L-NAME restored the inhibitory influence of thimerosal. The results indicate that NO and cysNO suppress baroreceptor activity through a mechanism independent of guanylate cyclase activation and vascular relaxation and that endogenous NO released by chemical activation suppresses baroreceptor activity.
Complement-Mediated Loss of Endothelium-Dependent Relaxation of Porcine Coronary Arteries
Authors: Stahl Gregory L. , Reenstra Wende R. , Frendl Gyorgy
Published: June 2012

Abstract
Reperfusion of the ischemic myocardium results in the loss of endothelium-dependent relaxation. We have shown recently that the alternate complement pathway is activated immediately on reperfusion of the ischemic porcine myocardium. We hypothesized that complement activation directly attenuates endothelium-dependent relaxation of porcine coronary arteries. Bradykinin (BK) or substance P concentration-dependently relaxed precontracted (U46619, 50 nmol/L) left anterior descending coronary artery (LAD) rings in vitro. Addition of zymosan to human (10%) or porcine (10%) serum for 30 minutes significantly (
P
<0.05) increased the EC
50
of BK-induced LAD relaxation from 4±1 to 418±159 nmol/L (n=8) and from 9±3 to 281±132 nmol/L (n=7), respectively. Similarly, addition of zymosan to 10% human serum (HS) for 30 minutes increased the EC
50
of substance P–induced LAD relaxation from 0.4±0.1 to 30±14 nmol/L (n=9,
P
<.05). Basal release of nitric oxide was reduced significantly in LAD rings exposed to zymosan-activated HS compared with HS alone. Addition of soluble CR1 (sCR1, 10 nmol/L) to zymosan-activated HS preserved BK-induced relaxation (EC
50
) of the LAD rings (control, 4±1 nmol/L; sCR1+zymosan+serum, 2±1 nmol/L; n=6). Zymosan-activated C8-depleted HS (10%) did not attenuate the EC
50
of BK-induced coronary artery relaxation (3±1 to 3±1 nmol/L, n=7,
P
=NS). Zymosan-activated C8-depleted HS plus C8 (6 μg/mL) increased the EC
50
of BK-induced coronary artery relaxation from 4±1 to 423±141 nmol/L (n=12,
P
<.05). We have further demonstrated that C5b-9 complexes can be found on the luminal surface of LAD endothelial cells after 5 minutes of exposure to zymosan-activated HS by using C5b-9 reactive monoclonal antibody fluorescent immunohistochemistry and confocal microscopy. We conclude that complement activation directly attenuates endothelium-dependent relaxation through the formation of the terminal membrane attack complex (C5b-9).
Conservation of an AE3 Cl
−
/HCO
3
−
Exchanger Cardiac-Specific Exon and Promoter Region and AE3 mRNA Expression Patterns in Murine and Human Hearts
Authors: Linn Stephen C. , Askew G. Roger , Menon Anil G. , Shull Gary E.
Published: June 2012

Abstract
A cardiac-specific variant of the rat AE3 Cl
−
/HCO
3
−
exchanger mRNA is transcribed from a promoter located in the sixth intron of a larger transcription unit expressed in brain and other tissues. The cardiac mRNA contains an alternative first exon encoding a 73–amino acid NH
2
-terminal sequence that replaces the first 270 amino acids of the brain AE3 variant. The present study was conducted to determine whether the cardiac-specific promoter region and exon are conserved in other species and to examine the expression patterns of AE3 mRNAs in adult tissues and during development. Analysis of murine and human genomic clones showed that both contain counterparts of the rat alternative exon. The cardiac promoter region is highly conserved in all three species and contains several potential transcription factor binding sites, including consensus MCAT and E-box sequences. Tissue-specific and developmental patterns of AE3 gene expression were examined by Northern blot hybridization. Mouse and human, like the rat, express both the 3.6-kb cardiac-specific AE3 mRNA and a 4.4-kb AE3 transcript found in brain, heart, and other tissues. Levels of the cardiac-specific transcript in mouse heart increase 17-fold between the fetal and adult stages, while the amount of the longer AE3 mRNA in heart decreases substantially. Furthermore, although the cardiac-specific mRNA is expressed in both atria and ventricles of mouse heart, the longer transcript is confined to the atria. These results suggest that the two AE3 variants have distinct roles in cardiac function and that the mechanisms regulating their expression are different.