bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitric oxide-mediated effects of interleukin-6 on [Ca2+]i and cell contraction in cultured chick ventricular myocytes.
Authors: Kinugawa K , Takahashi T , Kohmoto O , Yao A , Aoyagi T , Momomura S , Hirata Y , Serizawa T
Published: June 2012

Cytokines have significant roles in some cardiovascular disorders, but direct myocardial effects of cytokines remain to be elucidated. In the present study, we examined both the early and delayed effects of interleukin-6 (IL-6) on cultured chick embryo ventricular myocytes. Exposure of these cells to human recombinant IL-6 significantly decreased peak systolic [Ca2+]i (71.0 +/- 0.6% of the control value) and the amplitude of cell contraction (66.0 +/- 7.4% of the control value) within a few minutes. Pretreatment with NG-monomethyl-L-arginine (L-NMMA) or methylene blue completely inhibited the IL-6-induced early changes. Subsequent addition of L-arginine reversed the effects of L-NMMA. The levels of cGMP were significantly increased after 30 minutes of exposure to IL-6 (134.4 +/- 9.1% of the control value). Pretreatment with L-NMMA or EGTA significantly inhibited the IL-6-induced early elevation of cGMP. These results suggest that IL-6 acutely decreases intracellular Ca2+ transients and depresses cell contraction by nitric oxide (NO)-cGMP-mediated pathway. Therefore, IL-6 may enhance the Ca(2+)-dependent constitutive NO synthase activity in cardiac myocytes. On the other hand, 24-hour exposure to IL-6 also increased the levels of cGMP (159.0 +/- 22.8% of the control value) regardless of pretreatment with EGTA. These delayed increases in cGMP were also shown to be coupled with decreases in intracellular Ca2+ transients and the amplitude of cell contraction. Thus, IL-6 may induce Ca(2+)-independent NO synthase in cardiac myocytes. Together with the previous reports that have suggested the possible roles of IL-6 in myocardial stunning or endotoxic shock, this negative inotropic effect of IL-6 may contribute to these clinical settings.
alpha 1-adrenergic agonists precondition rabbit ischemic myocardium independent of adenosine by direct activation of protein kinase C.
Authors: Tsuchida A , Liu Y , Liu G S , Cohen M V , Downey J M
Published: June 2012

Ischemic preconditioning in the rabbit is initiated by adenosine A1-receptor stimulation, which activates protein kinase C (PKC). Additionally, alpha 1-adrenergic agonists can similarly protect ischemic myocardium, but there has been confusion about the role adenosine receptors play in this protection. To characterize the interaction between adrenergic and adenosine receptors and to study the possible role of PKC in this protection, we used isolated rabbit hearts perfused with oxygenated Krebs' buffer. All hearts were subjected to 30 minutes of regional myocardial ischemia and 2 hours of reperfusion. Infarct size was determined by triphenyltetrazolium staining. Pharmacologic preconditioning in hearts with a 5-minute phenylephrine (PE) infusion 10 minutes before the prolonged regional ischemia resulted in significantly smaller infarcts (9.7 +/- 1.3% of risk area) than in control hearts (31.0 +/- 2.6%, P < .05). This protection could be effectively blocked by administration of the alpha-adrenergic blocker phenoxybenzamine. Methoxamine, an alpha 1a-selective agonist, failed to protect, whereas the alpha 1b-selective antagonist chloroethylclonidine aborted the protective effect of PE. Polymyxin B, an inhibitor of PKC, also blocked the protective effect of PE, implying that PKC has an important role in preconditioning. The adenosine receptor blocker 8-(p-sulfophenyl)theophylline (SPT) given at the same time as the PE infusion did not affect the protection, implying that an alpha 1-agonist could initiate protection independent of adenosine, presumably by direct coupling to PKC. However, the protective effect of PE could be blocked if SPT were administered during the 30-minute regional ischemia. This observation suggested that adenosine receptor occupancy is necessary during long ischemia to reactivate PKC and mediate the protection. However, the addition of a second PE infusion beginning 5 minutes before and continuing throughout the long ischemic period restored the protective effect of PE despite the presence of SPT. Thus, as long as at least one of the receptors (alpha 1-adrenegic or adenosine A1) is activated during long ischemia, protection will be realized. These data indicate that alpha 1 receptors do not precondition through an adenosine intermediate but that alpha 1-adrenergic and adenosine receptors activate parallel pathways within the myocyte that can trigger and mediate protection.
Role of Ca2+ and protein kinase C in shear stress-induced actin depolymerization and endothelin 1 gene expression.
Authors: Morita T , Kurihara H , Maemura K , Yoshizumi M , Nagai R , Yazaki Y
Published: June 2012

Vascular endothelial cells adapt to changes in blood flow by altering the cell architecture and by producing various substances. We have previously reported that low shear stress induces endothelin 1 (ET-1) expression in endothelial cells and that this induction is mediated by depolymerization of actin fiber. In the present study, we examined the role of Ca2+ and protein kinase C (PKC) in shear stress-induced actin depolymerization and subsequent ET-1 gene expression. Exposure of cultured porcine aortic endothelial cells to low shear stress (5 dyne/cm2) for 3 hours increased the ratio of G-actin to total actin from 54 +/- 0.8% to 80 +/- 1.0%. This shear stress-induced actin depolymerization was completely blocked by chelation of extracellular Ca2+ with EGTA and partially inhibited by intracellular Ca2+ chelation with the tetraacetoxymethyl ester of BAPTA (BAPTA/AM). Pretreatment with staurosporine, a PKC inhibitor, or desensitization of PKC by treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) for 24 hours also resulted in partial inhibition of shear stress-induced actin depolymerization. Although PKC activation by TPA mildly increased G-actin content, the effect of TPA and shear stress on actin depolymerization was not additive. Moreover, shear stress-induced ET-1 gene expression was inhibited by EGTA, BAPTA/AM, and staurosporine to a degree similar to the inhibition of actin depolymerization. In contrast, ET-1 gene expression induced by cytochalasin B, an actin-disrupting agent, was not affected by staurosporine.(ABSTRACT TRUNCATED AT 250 WORDS)
Different functions of the platelet-derived growth factor-alpha and -beta receptors for the migration and proliferation of cultured baboon smooth muscle cells.
Authors: Koyama N , Hart C E , Clowes A W
Published: June 2012

Migration of medial smooth muscle cells (SMCs) and their proliferation in the intima contribute to thickening of injured and atherosclerotic vessels. These events have been proposed to be regulated in part by platelet-derived growth factor (PDGF). Two separate PDGF receptors have been identified, PDGF-R alpha and PDGF-R beta. To study the functions of PDGF-R alpha and PDGF-R beta in vascular SMCs, neutralizing monoclonal antibodies (mAbs) specific for each of the two receptors were used. These antibodies allowed us to evaluate the role of each receptor for PDGF-induced proliferation and migration of cultured baboon SMCs. Both PDGF-AA and PDGF-BB stimulated SMC growth, with PDGF-BB being more potent than PDGF-AA. Studies with anti-PDGF-R alpha and anti-PDGF-R beta mAbs revealed that both PDGF receptors promoted the stimulatory signals for proliferation. In contrast, PDGF-BB stimulated SMC migration, whereas PDGF-AA had no stimulatory activity on its own. Additionally, PDGF-AA was able to suppress migration induced by PDGF-BB or fibronectin in modified Boyden's chamber assay. When PDGF-BB-induced migration was separated into chemotactic and chemokinetic activities, only the chemotactic component was inhibited by PDGF-AA. The suppression of SMC migration by PDGF-AA was eliminated by anti-PDGF-R alpha mAb. In addition, PDGF-BB, in the presence of anti-PDGF-R beta, bound only to PDGF-R alpha and caused suppression of SMC migration induced by fibronectin. These results suggest that when activated by ligand binding, both PDGF-R alpha and PDGF-R beta stimulate proliferation. In contrast, only activation of PDGF-R beta stimulates migration, whereas ligand binding to PDGF-R alpha leads to inhibition of cell migration. These observations provide support for the conclusion that PDGF-R alpha and PDGF-R beta may play different roles in SMC function and may be involved in different regulatory mechanisms during vascular remodeling.
Different electrical responses to vasoactive agonists in morphologically distinct smooth muscle cell types.
Authors: Neylon C B , Avdonin P V , Dilley R J , Larsen M A , Tkachuk V A , Bobik A
Published: June 2012

Vascular smooth muscle cells (SMCs) in the blood vessel wall are frequently heterogeneous in nature, differing in their gross morphology, size, and shape, subcellular organelles, cytoskeleton, and contractile protein composition. In adult rat arterial vessels, two populations of SMCs have been shown to predominate: elongated bipolar cells, representing the majority of cells, and epithelial-like SMCs. We examined the ionic responses of these two types of SMCs, isolated by multiple subculture, to vasoactive stimuli. Elevations in intracellular Na+ and Ca2+ were measured with SBFI and fura 2, respectively, and changes in membrane potential were measured using the potential-sensitive fluorescent probe bis-oxonol. The resting membrane potential of the elongated bipolar cells was less negative than that of the epithelial-like SMCs. Exposure of the elongated SMCs to endothelin 1, alpha-thrombin, or arginine vasopressin induced elevations in [Ca2+]i and [Na+]i and membrane depolarization. Depolarization occurred because of entry of both Na+ and Ca2+, and pharmacological blockade of Cl- or K+ channels did not attenuate the depolarization. In contrast, when [Ca2+]i was elevated by the same agonists in the epithelial-like SMCs there was a pronounced hyperpolarization that appeared to be the consequence of enhanced activity of charybdotoxin-sensitive Ca(2+)-activated K+ channels because it was abolished by charybdotoxin (20 nmol/L), partially attenuated by tetraethylammonium chloride (10 mmol/L), and unaffected by apamin (1 mumol/L), glibenclamide (1 mumol/L), or 4-aminopyridine (5 mmol/L). Chelation of [Ca2+]i also abolished the hyperpolarization; instead, a small depolarization was observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Cardiac high-energy phosphates adapt faster than oxygen consumption to changes in heart rate.
Authors: Eijgelshoven M H , van Beek J H , Mottet I , Nederhoff M G , van Echteld C J , Westerhof N
Published: June 2012

To investigate the dynamic control of cardiac ATP synthesis, we simultaneously determined the time course of mitochondrial oxygen consumption with the time course of changes in high-energy phosphates following steps in cardiac energy demand. Isolated isovolumically contracting rabbit hearts were perfused with Tyrode's solution at 28 degrees C (n = 7) or at 37 degrees C (n = 7). Coronary arterial and venous oxygen tensions were monitored with fast-responding oxygen electrodes. A cyclic pacing protocol in which we applied 64 step changes between two different heart rates was used. This enabled nuclear magnetic resonance measurement of the phosphate metabolites with a time resolution of approximately 2 seconds. Oxygen consumption changed after heart-rate steps with time constants of 14 +/- 1 (mean +/- SEM) seconds at 28 degrees C and 11 +/- 1 seconds at 37 degrees C, which are already corrected for diffusion and vascular transport delays. Doubling of the heart rate resulted in a significant decrease in phosphocreatine (PCr) content (11% at 28 degrees C, 8% at 37 degrees C), which was matched by an increase in inorganic phosphate (P(i)) content, although oxygen supply was shown to be nonlimiting. The time constants for the change of both P(i) and PCr content, approximately 5 seconds at 28 degrees C and 2.5 seconds at 37 degrees C, are significantly smaller than the respective time constants for oxygen consumption.(ABSTRACT TRUNCATED AT 250 WORDS)
Properties of single outwardly rectifying Cl- channels in heart.
Authors: Duan D , Nattel S
Published: June 2012

A variety of potentially important macroscopic Cl- currents have been described in the heart. Although the single-channel properties of the cAMP-dependent current (ICl.cAMP) have been well described, the single-channel equivalents of the other forms of cardiac Cl- current remain unknown. Unlike ICl.cAMP, many of these currents show prominent outward rectification in the presence of symmetrical transmembrane Cl- gradients and sensitivity to disulfonic stilbene Cl- transport blockers. We used the patch-clamp technique to search for single Cl- channels in inside-out patches from rabbit atrial cell membranes, under conditions minimizing the chances of observing channels carrying Na+, Ca2+, or K+. Under symmetrical Cl- conditions, single-channel activity was seen in 14 (9%) of 155 patches. Channels showed strong outward rectification and a unitary conductance of 60 +/- 3 picosiemens (mean +/- SEM) at positive voltages. The current-voltage relation was not altered by replacement of cations by the impermeant cation N'-methyl-D-glucamine (NMDG) and shifted as expected for a Cl(-)-selective channel when methanesulfonate was substituted for Cl-. The Cl- transport blockers DIDS (diisothiocyanatostilbene-2,2'-disulfonic acid, 100 mumol/L) and SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, 1 mmol/L) strongly and reversibly inhibited channel activity when added to the bath and caused channel flickering suggesting open-channel block. Ensemble-average currents showed no time dependence, and the form of the ensemble-average current-voltage relation was similar to that of macroscopic background Cl- current.(ABSTRACT TRUNCATED AT 250 WORDS)
Multifunctional Ca2+/calmodulin-dependent protein kinase mediates Ca(2+)-induced enhancement of the L-type Ca2+ current in rabbit ventricular myocytes.
Authors: Anderson M E , Braun A P , Schulman H , Premack B A
Published: June 2012

The intracellular mechanism underlying the Ca(2+)-induced enhancement of the L-type Ca2+ current (ICa) was examined in adult rabbit cardiac ventricular myocytes by using patch-clamp methodology. Internal Ca2+ was elevated by flash photolysis of the Ca2+ chelator Nitr 5, and intracellular Ca2+ levels were simultaneously monitored by Fluo 3 fluorescence. Flash photolysis of Nitr 5 produced a rapid (< 1-second) elevation of internal Ca2+, which led to enhancement (39% to 51% above control) of the peak inward Ca2+ current after a delay of 20 to 120 seconds. Internal dialysis of myocytes with synthetic inhibitory peptides derived from the pseudosubstrate (peptide 273-302) and calmodulin binding (peptide 291-317) regions within the regulatory domain of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) blocked enhancement of ICa produced by elevation of internal Ca2+ but not that produced by beta-adrenergic stimulation. These inhibitory peptides also had no effect on the elevation of internal Ca2+ produced by flash photolysis of Nitr 5. A pseudosubstrate inhibitory peptide derived from protein kinase C had no significant effect on Ca(2+)-dependent enhancement of ICa. We conclude that CaM kinase mediates the Ca(2+)-induced enhancement of ICa in mammalian cardiac myocytes by a mechanism likely involving direct phosphorylation of the L-type Ca2+ channel complex or an associated regulatory protein.
Block of IKs, the slow component of the delayed rectifier K+ current, by the diuretic agent indapamide in guinea pig myocytes.
Authors: Turgeon J , Daleau P , Bennett P B , Wiggins S S , Selby L , Roden D M
Published: June 2012

There is a high incidence of diuretic use among patients who develop exaggerated QT prolongation and polymorphic ventricular tachycardia (torsade de pointes) during treatment with action potential-prolonging agents. Diuretic-induced hypokalemia is thought to be the usual mechanism, but a direct effect of diuretic drugs on repolarizing currents is an additional possibility. Therefore, in this study, we examined the effects of the diuretic agents chlorthalidone and indapamide on the cardiac delayed rectifier current. In guinea pig ventricular myocytes, this current is made up of two components: IKr, a rapidly activating, inwardly rectifying current blocked by most action potential-prolonging antiarrhythmics, and IKs, a slowly activating component. In this preparation, indapamide blocked outward current in a time-, voltage- and concentration-dependent fashion, whereas chlorthalidone (1 mmol/L) was without effect. The following features of the effect of indapamide strongly suggest selective block of IKs: (1) Indapamide block was significantly greater with 5000-millisecond activating pulses (-43 +/- 5% at +50 mV [100 mumol/L indapamide]) than with 225-millisecond ones (-20 +/- 5%; n = 5, P < .01), and the signature of the indapamide-sensitive current was a slowly activating delayed rectifier current. (2) The voltage dependence of indapamide block (EC50, 101 mumol/L at +50 mV and 196 mumol/L at +10 mV) was consistent with preferential block of IKs relative to IKr. (3) In the presence of indapamide, an envelope-of-tails test for IKr was satisfied. The drug-insensitive current had rectifying properties similar to those described for IKr in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Resistance to blood flow in microvessels in vivo.
Authors: Pries A R , Secomb T W , Gessner T , Sperandio M B , Gross J F , Gaehtgens P
Published: June 2012

Resistance to blood flow through peripheral vascular beds strongly influences cardiovascular function and transport to tissue. For a given vascular architecture, flow resistance is determined by the rheological behavior of blood flowing through microvessels. A new approach for calculating the contribution of blood rheology to microvascular flow resistance is presented. Morphology (diameter and length), flow velocity, hematocrit, and topological position were determined for all vessel segments (up to 913) of terminal microcirculatory networks in the rat mesentery by intravital microscopy. Flow velocity and hematocrit were also predicted from mathematical flow simulations, in which the assumed dependence of flow resistance on diameter, hematocrit, and shear rate was optimized to minimize the deviation between measured and predicted values. For microvessels with diameters below approximately 40 microns, the resulting flow resistances are markedly higher and show a stronger dependence on hematocrit than previously estimated from measurements of blood flow in narrow glass tubes. For example, flow resistance in 10-microns microvessels at normal hematocrit is found to exceed that of a corresponding glass tube by a factor of approximately 4. In separate experiments, flow resistance of microvascular networks was estimated from direct measurements of total pressure drop and volume flow, at systemic hematocrits intentionally varied from 0.08 to 0.68. The results agree closely with predictions based on the above-optimized resistance but not with predictions based on glass-tube data. The unexpectedly high flow resistance in small microvessels may be related to interactions between blood components and the inner vessel surface that do not occur in smooth-walled tubes.
The anti-inflammatory agent alpha-trinositol exerts its edema-preventing effects through modulation of beta 1 integrin function.
Authors: Rodt S A , Reed R K , Ljungström M , Gustafsson T O , Rubin K
Published: June 2012

Edema formation in acute inflammation can be induced through lowering of interstitial fluid pressure (Pif) and seems to involve dynamic beta 1 integrin-mediated interactions between dermal cells and extracellular matrix fibers. The present experiments investigate the role of beta 1 integrins in the control of Pif. The anti-inflammatory drug alpha-trinositol (1,2,6-D-myo-inositol trisphosphate) stabilizes Pif in acute inflammation. Pretreatment with 5 mg IV alpha-trinositol in pentobarbital-anesthetized rats inhibited the lowering in Pif and the edema formation induced by subdermal injection of anti-beta 1 integrin IgG. This stabilization of the beta 1 integrin function in vivo was paralleled by effects of alpha-trinositol on contraction of fibroblast-populated three-dimensional collagen lattices in vitro. alpha-Trinositol was additive to the known stimulatory effect of platelet-derived growth factor-BB on the final gel size in the collagen gel contraction assay. Furthermore, alpha-trinositol counteracted the inhibitory effect of anti-beta 1 integrin Fab fragments on collagen gel contraction. Finally, subdermal injection of dibutyryl-cAMP (db-cAMP) induced increased negativity of Pif to the same extent as did anti-beta 1 integrin antibodies, and in vitro db-cAMP reduced the ability of fibroblasts to contract collagen gels. The latter effect was opposed by alpha-trinositol. The data demonstrate that alpha-trinositol modulates beta 1 integrin function and may do so via intracellular pathways in turn affecting the function and/or cell surface expression of beta 1 integrins and suggest that alpha-trinositol can serve as a tool to study integrin function. Furthermore, the data indicate that the collagen contraction assays may provide important information of the control of Pif in vivo.
Substrate competition in postischemic myocardium. Effect of substrate availability during reperfusion on metabolic and contractile recovery in isolated rat hearts.
Authors: Tamm C , Benzi R , Papageorgiou I , Tardy I , Lerch R
Published: June 2012

Normal myocardium can derive energy for contraction and relaxation from oxidative metabolism of a variety of substrates. This investigation examined the influence of substrate availability early during reperfusion on the substrate pattern of oxidative metabolism and recovery of contractile function. For this purpose, isovolumically beating isolated rat hearts, perfused retrogradely with erythrocyte-supplemented buffer containing 0.4 mmol/L palmitate and 11 mmol/L glucose, were subjected to 40 minutes of no-flow ischemia. Hearts were reperfused with medium containing selected concentrations of palmitate and glucose. The substrate pattern for oxidative metabolism was determined on the basis of myocardial release of 14CO2 after equilibration of the hearts during the initial 15 minutes of reperfusion with either [1-14C]palmitate or [U-14C]glucose. In continuously perfused control hearts, glucose oxidation was largely inhibited by palmitate. During postischemic reperfusion, oxidation of glucose was increased by 59% (P < .05) and 467% (P <.01) in hearts reperfused after the ischemic period with 11 mmol/L glucose plus 0.4 or 1.2 mmol/L palmitate, respectively. Oxidation of palmitate was concomitantly reduced during reperfusion at low (0.4 mmol/L) but not at high (1.2 mmol/L) palmitate concentration. Compared with hearts reperfused with medium containing 0.4 mmol/L palmitate as sole substrate, hearts reperfused with medium containing 11 mmol/L glucose with 0.4 mmol/L palmitate exhibited lower left ventricular diastolic pressure (69 +/- 5 versus 90 +/- 3 mm Hg [mean +/- SEM], P < .05), less release of creatine kinase (31 +/- 5 versus 59 +/- 7 U/g wet wt, P < .05), and better recovery of left ventricular pressure development (26 +/- 9 versus 6 +/- 4 mm Hg, P < .05). Omission of palmitate or increasing the palmitate concentration to 1.2 mmol/L did not significantly alter postischemic myocardial contracture and enzyme release. The findings support the view that glucose oxidation early during reperfusion may be crucial for functional recovery. The results further indicate that interaction of substrates of oxidative metabolism is altered in severely injured postischemic myocardium. Inhibition of glucose oxidation by fatty acids was partially reversed during reperfusion.
Growth-promoting effects of substance P on endothelial cells in vitro. Synergism with calcitonin gene-related peptide, insulin, and plasma factors.
Authors: Villablanca A C , Murphy C J , Reid T W
Published: June 2012

The purpose of this study was to determine the effects of the vasoactive perivascular neuropeptide substance P (SP) on the growth and function of vascular endothelial cells in serum-free culture conditions with cells quiescent in the G0-G1 phase of the cell cycle and to characterize the response. In addition, interactions between SP and other growth factors and neuropeptides including insulin, platelet factors, neurokinin A, neurokinin B, and calcitonin gene-related peptide (CGRP) were studied on endothelial cell growth and compared. Growth effects were determined by stimulation of tritiated thymidine incorporation into DNA and cell proliferation. SP exhibited differential effects on cell growth that were a function of concentration, incubation time, interaction with other growth factors, and cell culture conditions. DNA synthesis in response to SP showed a bell-shaped distribution with a maximal effect that was 10.5-fold over control at 500 micrograms/mL of SP after 48 hours of incubation. The effect showed marked synergism with insulin (10 micrograms/mL) and with CGRP (0.01 to 10 micrograms/mL), which is colocalized with SP in vivo. Insulin and CGRP alone had no significant effect on endothelial cell growth. Furthermore, no synergism was observed between SP and platelet-derived growth factor or platelet-derived endothelial cell growth factor. Endothelial cell proliferation increased in response to SP to 2.6-fold over control at 48 hours, was maximal at 10 micrograms/mL SP, and also demonstrated synergism with insulin (10 micrograms/mL). Our studies indicate that neuropeptides play a significant role in regulating endothelial cell growth and proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)
Involvement of Na(+)-H+ antiporter in regulation of L-type Ca2+ channel current by angiotensin II in rabbit ventricular myocytes.
Authors: Kaibara M , Mitarai S , Yano K , Kameyama M
Published: June 2012

The present study investigated the possible involvement of a Na(+)-H+ antiporter in the regulation of L-type Ca2+ channels by angiotensin II (Ang II) in isolated rabbit ventricular cardiac myocytes by using both cell-attached and whole-cell patch-clamp current recording techniques. In cell-attached patch-clamp current recordings, an increase in the open-state probability of the Ca2+ channel (144.8 +/- 9.8% [mean +/- SEM], n = 11) was seen after exposure of the cells to Ang II (100 nmol/L). This effect was inhibited by pretreatment with losartan (10 mumol/L), a synthetic antagonist of the AT1 receptor. 5(N,N-Dimethyl)amiloride (100 mumol/L), an amiloride analogue, as well as Na(+)-deficient bath solution abolished Ang II-induced stimulation of the Ca2+ channel activities. In whole-cell patch-clamp current recordings, Ang II also increased the L-type Ca2+ current when a pipette solution of pH 7.1 containing 5 mmol/L HEPES (139 +/- 5%, n = 4) was used but did not significantly increase the current when a pipette solution of pH 7.5 containing 5 mmol/L HEPES or a pipette solution of pH 7.1 containing 30 mmol/L HEPES was used. These results suggest that Ang II-induced stimulation of the Ca2+ channels is mediated by a Na(+)-H+ antiporter and therefore provide a novel insight into signal transduction of Ang II receptor stimulation in cardiac myocytes.
Angiotensin II and Other Hypertrophic Stimuli Mediated by G Protein–Coupled Receptors Activate Tyrosine Kinase, Mitogen-Activated Protein Kinase, and 90-kD S6 Kinase in Cardiac Myocytes
Authors: Sadoshima Junichi , Qiu Zhihua , Morgan James P. , Izumo Seigo
Published: June 2012

Abstract
Many hypertrophic stimuli such as angiotensin II (Ang II) activate phospholipases through G protein–coupled receptors in cardiac myocytes. However, it is not known whether these stimuli also activate the tyrosine phosphorylation–dependent signaling pathway, which plays an essential role in growth factor–induced mitogenic responses in other cell types. Serine/threonine kinases such as mitogen-activated protein (MAP) kinases and 90-kD S6 kinase (RSK) are activated in response to many growth stimuli and are important downstream signaling pathways of tyrosine kinases. Therefore, we examined whether Ang II activates these protein kinases in primary cultures of cardiac myocytes and fibroblasts from neonatal rats. Ang II rapidly induced tyrosine phosphorylation of multiple proteins, including 42-, 44-, 75- to 80-, and 120- to 130-kD proteins, in both cardiac myocytes and fibroblasts. This was accompanied by an increase in tyrosine kinase activity. The 42- and 44-kD proteins were immunologically related to an extracellular signal-regulated kinase family (MAP kinases). Ang II rapidly increased kinase activity of MAP kinases and their downstream kinase, RSK. The Ang II–induced tyrosine phosphorylation and activation of MAP kinases and RSK were AT
1
receptor–mediated. Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate or an increase in intracellular Ca
2+
by the Ca
2+
ionophore A23187 was sufficient to cause tyrosine phosphorylation of multiple proteins and activation of MAP kinase and RSK. Although downregulation of PKC did not suppress Ang II–induced activation of MAP kinase and RSK, chelating intracellular Ca
2+
by BAPTA-AM completely abolished Ang II–induced activation of these kinases. Activation of MAP kinases and RSK was also observed in myocytes stimulated with other agonists for G
q
protein–coupled receptors, such as phenylephrine, norepinephrine, and endothelin 1, but not with agonists to G
s
protein–coupled receptors, such as isoproterenol. These results suggest that Ang II and other hypertrophic stimuli, known to act through G
q
protein–coupled receptors, rapidly cause tyrosine phosphorylation of several intracellular substrates through activation of tyrosine kinase and activate MAP kinases and RSK in cardiac myocytes as well as in cardiac fibroblasts. Furthermore, intracellular Ca
2+
, rather than PKC, seems to be critical for Ang II–induced activation of these protein kinases in cardiac myocytes.
Inhibition and Rapid Recovery of Ca
2+
Current During Ca
2+
Release From Sarcoplasmic Reticulum in Guinea Pig Ventricular Myocytes
Authors: Sipido Karin R. , Callewaert Geert , Carmeliet Edward
Published: June 2012

Abstract
We have investigated the modulation of the L-type Ca
2+
channel by Ca
2+
released from the sarcoplasmic reticulum (SR) in single guinea pig ventricular myocytes under whole-cell voltage clamp. [Ca
2+
]
i
was monitored by fura 2. By use of impermeant monovalent cations in intracellular and extracellular solutions, the current through Na
+
channels, K
+
channels, nonspecific cation channels, and the Na
+
-Ca
2+
exchanger was effectively blocked. By altering the amount of Ca
2+
loading of the SR, the time course of the Ca
2+
current (I
Ca
) could be studied during various amplitudes of Ca
2+
release. In the presence of a large Ca
2+
release, fast inhibition of I
Ca
occurred, whereas on relaxation of [Ca
2+
]
i
, fast recovery was observed. The time course of this transient inhibition of I
Ca
reflected the time course of [Ca
2+
]
i
. However, the inhibition seen in the first 50 ms, ie, the time of net Ca
2+
release from the SR, exceeded the inhibition observed later during the pulse, suggesting the existence of a higher [Ca
2+
] near the channel during this time. Transient inhibition of I
Ca
during Ca
2+
release was observed to a similar degree at all potentials. It could still be observed in the presence of intracellular ATP-γ-S and of cAMP. Therefore, we conclude that the modulation of I
Ca
by Ca
2+
release from the SR is not related to dephosphorylation. It could be related to a reduction in the driving force and to a direct inhibition of the channel by [Ca
2+
]
i
. The observation that the degree of inhibition does not depend on membrane potential suggests that the Ca
2+
binding site for this modulation is located outside the pore. The transient nature of the modulation of I
Ca
by Ca
2+
release will contribute to the recovery of I
Ca
during prolonged action potentials.
Patterns of Capillary Plasma Perfusion in Brains of Conscious Rats During Normocapnia and Hypercapnia
Authors: Abounader Roger , Vogel Johannes , Kuschinsky Wolfgang
Published: June 2012

Abstract
The present study aimed to investigate the distribution pattern of plasma flow velocities in brain capillaries. We tested the hypothesis that plasma flow velocities are heterogeneous in the brain capillaries of normocapnic conscious rats and become more homogeneous during increased cerebral blood flow induced by hypercapnia. We developed a method that makes it possible to detect the distribution pattern of plasma flow velocities from the intravascular dye concentrations measured in different capillaries. Evans blue was injected intravenously as a bolus, and 3 to 4 seconds later the rats were decapitated. During this period, a steep increase in arterial dye concentration was verified by frequent arterial blood sampling. Under such conditions, divergent plasma flow velocities in different capillaries yield unequal intravascular dye concentrations. Dye concentrations were measured in several hundred capillaries of brain cryosections using quantitative fluorescence microscopy based on calibration curves obtained from anesthetized rats. The results show a high degree of variation in the intravascular dye concentration during normocapnia. During increasing stages of hypercapnia, the variation was gradually reduced. The coefficient of variation (SD/mean · 100) of intracapillary dye concentration decreased from 76% at normocapnia to 22% at extreme hypercapnia (P
co
2
of 87 mm Hg), thus showing an inverse correlation with arterial P
co
2
(
r
=.97). The heterogeneity of intravascular dye concentrations observed in the present experiments indicates heterogeneous velocities of plasma perfusion in different brain capillaries during normocapnia and a more homogeneous distribution pattern during hypercapnic hyperemia.
ATP-Sensitive K
+
Channels Mediate α
2D
-Adrenergic Receptor Contraction of Arteriolar Smooth Muscle and Reversal of Contraction by Hypoxia
Authors: Tateishi Jun , Faber James E.
Published: June 2012

Abstract
Evidence in rat skeletal muscle suggests that local metabolic control of blood flow is facilitated by the reliance on α
2D
-adrenergic receptors (ARs) for constriction of arterioles, together with the strong sensitivity of this constriction to inhibition by hypoxia. The present study examined the role of ATP-sensitive K
+
(K
ATP
) channels in the selective interaction between α
2D
-ARs and hypoxia. Arterioles from rat cremaster muscle that possess both α
1D
(α
1A/D
)- and α
2D
-AR subtypes were microcannulated, pressurized, and isolated in a tissue bath for measurement of changes in lumen diameter. Three studies first examined whether stimulation of α
2D
- and α
1D
-ARs involves inhibition of the K
ATP
channel. Concentration-dependent constriction by the K
ATP
antagonists glibenclamide (GLB, 0.01 to 10 μmol/L) and disopyramide (0.001 to 1 mmol/L) were abolished during α
2D
stimulation but unaffected during α
1D
stimulation. Activation of the K
ATP
channel by cromakalim inhibited α
2D
constriction with greater potency than α
1D
(EC
50
, 7.0±0.2 versus 6.3±0.1). Finally, GLB (0.5 μmol/L) abolished dose-dependent α
2D
constriction, whereas α
1D
was unaffected. These data suggest that α
2D
but not α
1D
stimulation is “coupled” with closure of the K
ATP
channel, leading to depolarization and contraction of vascular smooth muscle. In a second series, hypoxic (P
o
2
, 6 mm Hg) inhibition of intrinsic smooth muscle tone was completely reversed by 0.1 μmol/L GLB, concentration-dependent GLB constriction was enhanced during hypoxia, and hypoxia reversed GLB constriction. These data confirm reports by others that hypoxia potentiates the activation of K
ATP
channels, leading to hyperpolarization and relaxation. Finally, GLB constriction, which was abolished by concomitant α
2D
stimulation, was completely restored by simultaneous activation of K
ATP
channels with hypoxia. These findings suggest that the sensitivity of α
2D
-AR constriction to inhibition by hypoxia arises through “antagonistic coupling” between these two stimuli, by which the α
2D
-AR inhibits and hypoxia activates K
ATP
channels.
Preconditioning of Isolated Rat Heart Is Mediated by Protein Kinase C
Authors: Mitchell Max B. , Meng Xianzhong , Ao Lihua , Brown James M. , Harken Alden H. , Banerjee Anirban
Published: June 2012

Abstract
Catecholamines have been implicated in the phenomenon of ischemic preconditioning. We have previously demonstrated that ischemic preconditioning against postischemic mechanical dysfunction in the isolated rat heart is mediated by the α
1
-adrenergic receptor. The purpose of this study was to delineate the signal transduction of preconditioning distal to the α
1
-adrenergic receptor. Our results suggest that (1) transient ischemia and α
1
-adrenergic receptor–induced preconditioning is inhibited by protein kinase C (PKC) antagonists, (2) functional protection against global ischemia/reperfusion injury can be induced by infusion of diacylglycerol, the second messenger of the α
1
-adrenergic pathway, and (3) transient ischemia and α
1
-adrenergic preconditioning are both characterized by similar translocation of PKC-δ to the sarcolemma of myocardium. These findings suggest that PKC is an effector of preconditioning in the isolated rat heart.
Expression of Vascular Endothelial Growth Factor From a Defective Herpes Simplex Virus Type 1 Amplicon Vector Induces Angiogenesis in Mice
Authors: Mesri Enrique A. , Federoff Howard J. , Brownlee Michael
Published: June 2012

Abstract
Vascular endothelial growth factor (VEGF) is a secreted endothelial cell–specific angiogenic growth factor. VEGF gene transfer strategies to stimulate focal angiogenesis could be used to ameliorate myocardial ischemia. To induce angiogenesis in vivo, we have constructed a replication-defective herpes simplex virus type 1 (HSV-1) amplicon vector that places the human VEGF-165 cDNA under the transcriptional control of the HSV immediate-early 4/5 promoter (HSVhvegf). Transduction of NIH 3T3 fibroblasts with HSVhvegf resulted in the secretion of high levels of biologically active VEGF, as assayed by microvascular endothelial mitogenesis. By use of an ex vivo protocol, BLK-CL4 fibroblasts were transduced with HSVhvegf or control HSVlac virus (expressing
Escherichia coli
β-galactosidase), resuspended in basement membrane extract (matrigel), and coinjected subcutaneously into syngeneic C57BL/6 mice. One week later, the matrigel plugs with HSVhvegf showed a strong angiogenic response, in contrast to the plugs with HSVlac-transduced fibroblasts. These data indicate that transduction with HSVhvegf virus can induce an angiogenic response in vivo and suggest that this is a viable gene therapy approach for tissue ischemia.
c-
myc
in Vasculoproliferative Disease
Authors: Edelman Elazer R. , Simons Michael , Sirois Martin G. , Rosenberg Robert D.
Published: June 2012

Abstract
Antisense oligonucleotides to genes central to cellular proliferation have suppressed smooth muscle cell growth in vitro and in vivo. We now report that although the response of cultured smooth muscle cells to antisense oligonucleotides to c-
myc
and c-
myb
is identical, the response of the injured arterial wall to these oligomers depends on the kinetics of gene expression and oligonucleotide delivery. Two different antisense oligonucleotides to each oncogene were administered to the perivascular aspect of injured rat carotid arteries via polymer-based delivery systems. The acute release of antisense oligonucleotides from the Pluronic gels reduced in vitro cell growth 54.8% with c-
myc
and 56.9% with c-
myb
. The more sustained release from ethylene vinyl acetate copolymer (EVAc) matrices was slightly less efficient, inhibiting proliferation 47.3% and 43.3%, respectively. However, although both EVAc and Pluronic release of c-
myb
antisense oligonucleotide sequences inhibited intimal hyperplasia 2 weeks after injury, only the more prolonged EVAc matrix release of antisense oligonucleotide to c-
myc
was effective. The failure of the short course of c-
myc
oligomer release from Pluronic gels stemmed from early successful suppression with late loss of regulation and not from inactivation of the antisense oligonucleotide within the polymeric gel. Within 24 hours of injury, Pluronic-based release of c-
myc
antisense oligomers reduced mRNA levels in the tunica media 2.5-fold and immunocytochemical identification of c-
myc
expression by 98.8%. As a result, the number of proliferating cells was decreased 6.5-fold 3 days after injury. One week after injury, however, the effect of Pluronic gel–released c-
myc
antisense was lost on both the number of cells expressing the oncogene (only 29.6% suppression) and the extent of intimal hyperplasia (intimal-to-medial area ratio, 0.35±0.02 versus 0.5±0.1 in control arteries). In contrast, EVAc release of the same sequences obliterated protein expression, maintaining a 99.6±0.7% inhibition for the duration of the experiment, and reduced intimal hyperplasia 2 weeks after injury 11.1-fold. We conclude that the effectiveness of the antisense approach to suppression of neointimal proliferation depends on the time course of expression of the target gene as well as the kinetics of oligonucleotide delivery.
cGMP and Nitric Oxide Modulate Thrombin-Induced Endothelial Permeability
Authors: Draijer Richard , Atsma Douwe E. , van der Laarse Arnoud , van Hinsbergh Victor W.M.
Published: June 2012

Abstract
Previous studies have demonstrated that cGMP and cAMP reduce the endothelial permeability for fluids and macromolecules when the endothelial permeability is increased by thrombin. In this study, we have investigated the mechanism by which cGMP improves the endothelial barrier function and examined whether nitric oxide (NO) can serve as an endogenous modulator of endothelial barrier function. Thrombin increased the passage of macromolecules through human umbilical vein and human aortic endothelial cell monolayers and concomitantly increased [Ca]
2+
in vitro. Inhibition of these increases by the intracellular Ca
2+
chelator BAPTA indicated that cytoplasmic Ca
2+
elevation contributes to the thrombin-induced increase in endothelial permeability. The cGMP-dependent protein kinase activators 8-bromo-cGMP (8-Br-cGMP) and 8-(4-chlorophenylthio)cGMP (8-PCPT-cGMP) decreased the thrombin-induced passage of macromolecules. Two pathways accounted for this observation. Activation of cGMP-dependent protein kinase by 8-PCPT-cGMP decreased the accumulation of cytoplasmic Ca
2+
in aortic endothelial cells and hence reduced the thrombin-induced increase in permeability. On the other hand, in umbilical vein endothelial cells, cGMP-inhibited phosphodiesterase (PDE III) activity was mainly responsible for the cGMP-dependent reduction of endothelial permeability. The PDE III inhibitors Indolidan (LY195115) and
SKF94120
decreased the thrombin-induced increase in permeability by 50% in these cells. Thrombin treatment increased cGMP formation in the majority of, but not all, cell cultures. Inhibition of NO production by
N
G
-nitro-
l-
arginine methyl ester (L-NAME) enhanced the thrombin-induced increase in permeability, which was restricted to those cell cultures that displayed an increased cGMP formation after addition of thrombin. Simultaneous elevation of the endothelial cGMP concentration by atrial natriuretic factor, sodium nitroprusside, or 8-Br-cGMP prevented the additional increase in permeability induced by L-NAME. These data indicate that cGMP reduces thrombin-induced endothelial permeability by inhibition of the thrombin-induced Ca
2+
accumulation and/or by inhibition of cAMP degradation by PDE III. The relative contribution of these mechanisms differs in aortic and umbilical vein endothelial cells. NO can act in vitro as an endogenous permeability-counteracting agent by raising cGMP in endothelial cells of large vessels.
Adenosine A
2a
and A
2b
Receptors in Cultured Fetal Chick Heart Cells
Authors: Liang Bruce T. , Haltiwanger Brett
Published: June 2012

Abstract
Adenosine exerts pronounced biological effects in the heart cell. The role of multiple adenosine receptor subtypes in regulating the heart cell function is not known. Ventricular cells cultured from chick embryos 14 days in ovo were used to study a novel feature of heart cell regulation by the stimulatory adenosine receptors. The inhibitory adenosine A
1
receptor pathway was first inactivated by pertussis toxin treatment of the cultures, and the effects of adenosine agonists and antagonists on the heart cell contractile amplitude, measured via an opticovideo motion detection system, and on the modulation of cAMP level were determined. Adenosine and
N
-ethyladenosine-5′-uronic acid (NECA), capable of activating both the adenosine A
2a
and A
2b
receptors, caused a greater increase in the contractile amplitude than did the A
2a
-selective agonist 2-[4-(2-carboxyethyl)phenylethylamino]-5′-
N
-ethylcarboxamidoadenosine (
CGS21680
). NECA caused a biphasic increase in cAMP, which became monophasic in the presence of the A
2a
receptor–selective antagonist 8-(3-chlorostyryl)caffeine, whereas the
CGS21680
-induced cAMP response was monophasic. Blocking with 8-(3-chlorostyryl)caffeine abolished most of the
CGS21680
-elicited contractile or cAMP response while attenuating only part of the adenosine- or NECA-stimulated responses. Blocking with the A
2b
-selective antagonists 1,3-diethyl-8-phenylxanthine or alloxazine caused a more pronounced inhibition of the contractile or cAMP response by adenosine or NECA than by
CGS21680
. Affinity of the A
2a
receptor was 60-fold higher than that of the A
2b
receptor. These data demonstrate that a functional A
2b
receptor is expressed on the heart cell and is capable of mediating augmentation of cardiac myocyte contractility and that adenosine A
2a
and A
2b
receptors, with greatly different affinity, coexist and are coupled to the same functional responses. Taken together, the data suggest a novel feature of heart cell regulation, where the high-affinity A
2a
receptor can play an important modulatory role in the presence of a low level of adenosine, whereas the low-affinity A
2b
receptor becomes functionally important when the adenosine level is high.