bluuebunny/crossref_metadata_2025 huggingface dataset:
Angiotensin II stimulates two myelin basic protein/microtubule-associated protein 2 kinases in cultured vascular smooth muscle cells.
Authors: Tsuda T , Kawahara Y , Ishida Y , Koide M , Shii K , Yokoyama M
Published: June 2012

In cultured vascular smooth muscle cells, angiotensin II (Ang II) stimulated a cytosolic protein kinase activity toward myelin basic protein (MBP) in a time- and dose-dependent manner. Phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate also increased the MBP kinase activity. Downregulation of protein kinase C by prolonged treatment of the cells with phorbol 12,13-dibutyrate markedly attenuated the Ang II- and PMA-induced MBP kinase activation. The Ang II- and PMA-stimulated MBP kinase activities were resolved almost equally into two distinct fractions on Mono-Q HR5/5 column chromatography (kinase 1 and kinase 2). The kinase assay in polyacrylamide gel revealed that apparent molecular masses of kinase 1 and kinase 2 were 40 and 45 kd, respectively. Microtubule-associated protein 2 also served as a substrate for both the kinases. Immunoblot analysis with an antiphosphotyrosine antibody suggested that both the kinases were tyrosine-phosphorylated during the action of Ang II. Phosphoamino acid analysis revealed that Ang II and PMA induced phosphorylation of both the kinases on serine/threonine as well as tyrosine residues. Phosphopeptide mapping patterns of kinase 1 and kinase 2 isolated from Ang II-stimulated cells were almost identical with those from PMA-stimulated cells. These results indicate that in vascular smooth muscle cells Ang II activates two species of MBP/microtubule-associated protein 2 kinases mainly through the protein kinase C-signaling pathway and suggest that tyrosine and serine/threonine phosphorylation may be involved in this process.
Captopril enhances intracellular calcium handling and beta-adrenergic responsiveness of myocardium from rats with postinfarction failure.
Authors: Litwin S E , Morgan J P
Published: June 2012

To examine the cellular mechanisms of contractile dysfunction in postinfarction heart failure, we studied the effects of beta-adrenergic receptor stimulation on contractile function and Ca2+i handling of noninfarcted papillary muscles from sham-operated (n = 17) and infarcted (n = 17) rats. Ca2+i transients measured with the bioluminescent protein aequorin and parameters of isometric contraction were recorded during graded isoproterenol stimulation. Developed tension and peak rate of tension rise were depressed (p less than 0.05) in muscles from infarcted rats at physiological and maximally stimulating [Ca2+]oS. The time to peak tension was prolonged in the muscles from the infarcted rats, corresponding with prolongation of the time to peak Ca2+i. In muscles from sham-operated rats, isoproterenol increased both the amplitude of the Ca2+i transient and the peak rate of tension rise. In contrast, the inotropic response to isoproterenol was severely blunted in the muscles from infarcted rats despite a large increase in the amplitude of the Ca2+i transient. Isoproterenol abbreviated the time course of the isometric twitch and the Ca2+i transient in both groups. These findings suggest that postinfarction heart failure may be related in part to decreased force-generating capacity of the myofilaments. Treatment with captopril for 5 weeks, beginning 1 week after infarction (n = 14), resulted in reduction of left ventricular filling pressures and partial normalization of myocardial contractility and Ca2+i handling. In addition, compared with muscles from untreated infarcted rats, muscles from the captopril-treated rats exhibited improved contractile responses to increasing [Ca2+]o or isoproterenol. The inotropic response to isoproterenol in muscles from all three groups of rats had a significant negative correlation (r = -0.64, p less than 0.0001) with left ventricular end-diastolic pressure measured in vivo. Thus, the defect in excitation-contraction coupling in rats with postinfarction heart failure may be partially normalized by chronic load reduction with an angiotensin converting enzyme inhibitor.
Leukotriene B4 mediates shear rate-dependent leukocyte adhesion in mesenteric venules.
Authors: Bienvenu K , Russell J , Granger D N
Published: June 2012

Previous studies have demonstrated that low shear rates promote leukocyte adherence to microvascular endothelium in postcapillary venules. The objective of this study was to determine whether an accumulation of inflammatory mediators such as platelet activating factor and leukotriene B4 is responsible for shear rate-dependent leukocyte-endothelial cell adhesion. Postcapillary venules (25-39 microns in diameter) in cat mesentery were studied by intravital microscopy. Venular wall shear rate was varied over a wide range by graded occlusion of the mesenteric artery. Red blood cell velocity, vessel diameter, leukocyte rolling velocity, and the numbers of rolling and adherent leukocytes were measured at each shear rate. In one series of experiments, shear rate-dependent leukocyte adherence was monitored at different superfusion rates (1.0 and 2.5 ml/min). At the lower superfusion rate, the number of adherent leukocytes was significantly higher at any given shear rate when compared with results obtained at the higher superfusion rate. This suggests that reduced washout of inflammatory mediators contributes to shear rate-dependent leukocyte adhesion. Pretreatment with different platelet activating factor receptor antagonists (WEB 2086 or WEB 2170) had no effect on the number of adherent leukocytes normally observed at lower shear rates, suggesting that platelet activating factor does not play a major role in this process. However, shear rate-dependent leukocyte adhesion was largely prevented by pretreatment with either a leukotriene B4 receptor antagonist (SC-41930) or a leukotriene synthesis inhibitor (L663,536). The results of this study indicate that a reduced washout of leukotriene B4 is responsible for the enhanced leukocyte adherence that occurs at low venular wall shear rates.
Effect of perfusate [Ca2+] on cardiac sarcoplasmic reticulum Ca2+ release channel in isolated rat hearts.
Authors: Abdelmeguid A E , Feher J J
Published: June 2012

The effect of perfusate [Ca2+] on the function of cardiac sarcoplasmic reticulum (CSR) was assessed by the oxalate-supported Ca2+ uptake rate of ventricular homogenates of isolated rat hearts maintained in a modified Langendorff preparation. The total Ca2+ pumping activity of the CSR was determined by using 20 microM ruthenium red or 625 microM ryanodine to close the CSR Ca2+ release channel. The homogenate Ca2+ uptake rate in the absence of ruthenium red or ryanodine decreased progressively with increasing perfusate [Ca2+] (25.7 +/- 1.2, 21.4 +/- 1.5, 17.2 +/- 1.1, and 16.3 +/- 1.2 [mean +/- SEM] nmol Ca2+.min-1.mg-1 for hearts perfused for 5 minutes with 0.2, 1.4, 2.8, and 5.6 mM Ca2+, respectively; p = 0.0001; n = 8). This depression was not observed when Ca2+ uptake was assayed in the presence of ryanodine or ruthenium red. Since the Ca2+ uptake in the presence of ryanodine or ruthenium red is determined by the Ca(2+)-ATPase, this result suggests that perfusion with varying [Ca2+] did not affect the Ca(2+)-ATPase. The observed decrease in Ca2+ uptake in the absence of ryanodine or ruthenium red is caused by an increased efflux through the ryanodine-sensitive Ca2+ release channel. When hearts perfused for 5 minutes with 0.2 or 5.6 mM Ca2+ were reperfused for 10 minutes with 1.4 mM Ca2+, homogenate Ca2+ uptake rates were restored to near control levels. These effects of perfusate Ca2+ were not direct effects, because changes in the [Ca2+] of the homogenization medium did not alter the homogenate Ca2+ uptake activity in either the presence or absence of ryanodine. The homogenate Ca2+ uptake rates were unaffected by prior active loading of the CSR with Ca2+. These results suggest a regulatory role of perfusate Ca2+ in increasing the open state of the ryanodine-sensitive Ca2+ release channel that is distinct from the beat-to-beat regulation of Ca2+ release from the CSR by Ca2+ (Ca(2+)-induced Ca2+ release).
Reperfusion arrhythmias in isolated perfused pig hearts. Inhomogeneities in extracellular potassium, ST and TQ potentials, and transmembrane action potentials.
Authors: Coronel R , Wilms-Schopman F J , Opthof T , Cinca J , Fiolet J W , Janse M J
Published: June 2012

We recorded direct current electrograms and local [K+]o at multiple sites and transmembrane potentials at selected sites during reperfusion after 5 minutes and 10 minutes of regional ischemia in isolated perfused pig hearts. After 10 minutes of ischemia, the incidence of ventricular fibrillation (VF) was 38%. At 80-90 seconds after reperfusion, [K+]o was 0.8 mM less than in normal tissue in half of the reperfused tissue, especially in the border zone. This was associated with TQ elevation of +4.5 mV and large peaked T waves. The latter was caused by an abrupt decrease of action potential duration in reperfused tissue, leading to a difference of up to 165 msec with normal tissue. Reperfusion VF started with a closely coupled ventricular premature beat. Activation block between reperfused and normal tissue permitted reentrant activation, leading to VF. Pretreatment with ryanodine (10(-6) M) and reperfusion with elevated [K+] (both of which prevent delayed afterdepolarizations) did not prevent closely coupled ventricular premature beats or VF. Five minutes of ischemia never caused VF. K+ depletion and TQ elevation in the reperfused zone was less frequent and smaller (-0.4 mM and 1.8 mV, respectively). Peaked T waves did not occur, and shortening of the action potential duration was less. We conclude that extracellular K+ depletion and marked action potential duration shortening in the reperfused tissue play a role in the genesis of reperfusion VF, which is caused by reentry. The closely coupled ventricular premature beat that initiates reentry is not caused by delayed afterdepolarizations but most likely by intramural reentry.
Endothelin-1 enhances calcium entry through T-type calcium channels in cultured neonatal rat ventricular myocytes.
Authors: Furukawa T , Ito H , Nitta J , Tsujino M , Adachi S , Hiroe M , Marumo F , Sawanobori T , Hiraoka M
Published: June 2012

Endothelin-1 (ET-1), a 21-amino acid vasoconstrictive peptide, increases intracellular Ca2+ level and has hypertrophic action on ventricular myocytes. To elucidate a possible role of Ca2+ entry through sarcolemmal Ca2+ channels on this ET-1 action, we examined effects of ET-1 on L-type (ICa,L) and T-type (ICa,T) Ca2+ currents in cultured neonatal rat ventricular myocytes using the patch-clamp technique. ET-1 at a concentration of 10 nM increased the maximum current density of ICa,T from -3.0 +/- 1.4 microA/cm2 in the control condition to -4.4 +/- 1.6 microA/cm2 (p < 0.01). Although the peak amplitude of ICa,L was decreased during ET-1 application (from -9.7 +/- 1.9 microA/cm2 in the control condition to -5.0 +/- 1.4 microA/cm2 [p < 0.01]), this magnitude of decrease in ICa,T (52 +/- 19%) was comparable to that of spontaneous "run-down" of ICa,L (47 +/- 26%). The enhancement of ICa,T by ET-1 was dose dependent; it was initiated as low as 0.32 nM, and the maximal response was attained at approximately 10 nM, with a half-maximal dose of 1.26 nM. The enhancement of ICa,T by ET-1 was antagonized by protein kinase C inhibitors staurosporine (0.2 microM) and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7, 20 microM) applied to the pipette solution. Extracellular application of tumor-promoting phorbol esters, phorbol 12,13-dibutyrate (PDBu) and 4 beta-phorbol 12-myristate 13-acetate, augmented ICa,T. PDBu (0.2 microM) increased the maximal current density of ICa,T from -4.2 +/- 0.5 microA/cm2 in the control condition to -5.5 +/- 1.0 microA/cm2 (p < 0.01). In the presence of H-7 (20 microM) in the pipette solution, PDBu failed to enhance ICa,T, and an inactive isomer of PDBu (4 alpha-phorbol 12,13-dibutyrate, 0.2 microM) did not augment ICa,T. Thus, ET-1 enhances Ca2+ entry through the sarcolemmal T-type Ca2+ channel, possibly through a pathway involving activation of protein kinase C. This ET-1 action may be involved in the rise of the intracellular Ca2+ level and may contribute to the induction of cardiac hypertrophy by ET-1.
HCO3(-)-dependent intracellular pH regulation in the premature myocardium.
Authors: Nakanishi T , Gu H , Seguchi M , Cragoe E J , Momma K
Published: June 2012

This study investigated developmental changes in Na(+)-H+ exchange and HCO3(-)-Cl- exchange activities in newborn and adult rabbit hearts. pHi was measured using the fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in isolated myocytes. Myocardial mechanical function was measured in the isolated ventricular preparation. Intracellular acidosis with normal pHo was induced by an NH4Cl (10 mM) prepulse technique. Upon removal of NH4Cl, pHi fell transiently and then recovered toward the control level. In the HCO3-/CO2-buffered solution, the rate of recovery of pHi in the newborn was greater than in the adult. In the HCO3-/CO2-buffered solution, 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of Na(+)-H+ exchange, inhibited the recovery of pHi completely in the adult. In the newborn, however, significant recovery of pHi was observed in the presence of EIPA. In the presence of both EIPA and 4-acetamido-4'-isothiocyanatostilbene-2',2'-disulfonic acid (SITS), an inhibitor of HCO3(-)-Cl- exchange, the recovery of pHi was not observed in the two age groups. In the HEPES-buffered solution that did not contain HCO3-/CO2, the rate of recovery of pHi after NH4Cl removal was similar in the two age groups. In the HEPES-buffered solution, the recovery of pHi was completely inhibited by EIPA in the two age groups. In the presence of EIPA in the HCO3-/CO2-buffered solution, contractile function decreased during acidosis after NH4Cl removal and did not recover in the adult. In the newborn, significant recovery of contractile function was observed after NH4Cl removal in the presence of EIPA. The recovery of mechanical function observed in the presence of EIPA in the newborn was inhibited by SITS. These data suggest that, although there is no developmental change in the Na(+)-H+ exchange activity, HCO3(-)-Cl- exchange is more active in the premature myocardium. The presence of the HCO3(-)-Cl- exchanger is important in maintaining myocardial contractile function during acidosis, especially when Na(+)-H+ exchange is inhibited and may partly explain the greater resistance of the premature myocardium to acidosis.
Regulation of Na,K-ATPase gene expression by thyroid hormone in rat cardiocytes.
Authors: Kamitani T , Ikeda U , Muto S , Kawakami K , Nagano K , Tsuruya Y , Oguchi A , Yamamoto K , Hara Y , Kojima T
Published: June 2012

Synthesis and activity of the enzymatic equivalent of the sodium pump, Na,K-ATPase, are regulated by thyroid hormone in responsive tissues. The purpose of this study was to determine whether triiodothyronine (T3) regulates the level of the messenger RNA (mRNA) coding for Na,K-ATPase alpha- and beta-subunits in the heart. The expression of Na,K-ATPase mRNAs in in vitro myocardial cells was directly assayed by Northern and slot blot hybridization using Na,K-ATPase alpha- and beta-isoform-specific cDNA probes. Exposure of cultured neonatal rat cardiocytes to 10(-8) M T3 resulted in 1) threefold to fourfold increase in alpha 1- and beta 1-mRNA accumulation, with a maximum elevation at 48 hours, 2) sevenfold increase in alpha 2-mRNA accumulation with a peak elevation at 72 hours, and 3) transient threefold increase in alpha 3-mRNA within the first 24 hours followed by a deinduction thereafter. The increase in alpha 1-mRNA accumulation by T3 occurred over the physiological T3 concentration range with an EC50 of 5 x 10(-10) M. This was associated with a twofold increase in alpha 1-subunit protein accumulation and an increase in Na,K-ATPase transport activity. The half-life of alpha 1-mRNA analyzed by actinomycin D chase was less than 3 hours and was not affected by T3. Transfection experiments with the luciferase reporter gene revealed that thyroid hormone response sequences are located within the 5'-flanking regions of each alpha-isoform gene.(ABSTRACT TRUNCATED AT 250 WORDS)
Optimization of retroviral vector-mediated gene transfer into endothelial cells in vitro.
Authors: Kahn M L , Lee S W , Dichek D A
Published: June 2012

Retroviral vector-mediated gene transfer into endothelial cells is relatively inefficient with transduction rates as low as 1-2% in vitro and even lower in vivo. To increase the efficiency of gene transfer into endothelial cells, we used retroviral vectors expressing beta-galactosidase and urokinase and measured endothelial cell transduction efficiencies with quantitative assays for beta-galactosidase and urokinase protein. We evaluated several techniques reported to improve the efficiency of retroviral transduction in vitro, including 1) extended periods of exposure to vector, 2) repeated exposures to vector, 3) maximization of the ratio of vector particles to endothelial cells by increasing the volume and concentration of vector particles or by decreasing the number of endothelial cells exposed, 4) cocultivation of endothelial cells with vector-producing cells, and 5) variation of the type and concentration of polycation used with the retroviral vector. Only the use of more concentrated (higher titer) vector-containing supernatant and the use of the polycation DEAE-dextran improved the efficiency of gene transfer into endothelial cells in vitro. In an optimized transduction protocol, a 60-second exposure to 1 mg/ml DEAE-dextran followed by a single 6-hour exposure to supernatant of a titer of 10(5)-10(6) colony-forming units/ml resulted in transduction efficiencies of 50-90% with both vectors. Decreasing the time of the supernatant exposure to 15 minutes permitted transduction efficiencies of 15-20% while significantly minimizing the duration of the transduction. Therefore, the optimized protocol allows high efficiency in vitro gene transfer into endothelial cells within several hours. The briefer protocol may prove useful for in vivo gene transfer in which the time of exposure to the supernatant is limited.
Nitric oxide synthase in cardiac nerve fibers and neurons of rat and guinea pig heart.
Authors: Klimaschewski L , Kummer W , Mayer B , Couraud J Y , Preissler U , Philippin B , Heym C
Published: June 2012

Participation of nitric oxide (NO) in the autonomic innervation of rat and guinea pig hearts was investigated by applying the NADPH diaphorase technique and immunohistochemistry with NO synthase antiserum. We present evidence that NO synthase is localized in cardiac ganglion cells and nerve fibers innervating the sinuatrial and atrioventricular nodes, the myocardium, local neurons, coronary arteries, and pulmonary vessels, suggesting an involvement of NO in neurogenic heart rate regulation, myocardial cell function, neuronal transmission in cardiac ganglia, and coronary as well as pulmonary vasodilation.
20-Hydroxyeicosatetraenoic acid is an endogenous vasoconstrictor of canine renal arcuate arteries.
Authors: Ma Y H , Gebremedhin D , Schwartzman M L , Falck J R , Clark J E , Masters B S , Harder D R , Roman R J
Published: June 2012

Recent studies have indicated that renal arteries can produce 20-hydroxyeicosatetraenoic acid (20-HETE) and suggest the potential involvement of a P450 metabolite of arachidonic acid in the myogenic activation of canine renal arteries. In the present study, the effects of 20-HETE on isolated canine renal arcuate arteries were studied. Administration of 20-HETE to the bath or the lumen at concentrations of 0.01-1 microM produced a graded reduction in the diameter of these vessels. In contrast, 19(R)-HETE was a vasodilator, whereas 19(S)-HETE was relatively inactive. The vasoconstrictor response to 20-HETE was not altered by the cyclooxygenase inhibitor indomethacin, endoperoxide/thromboxane receptor antagonist SQ29548, or combined blockade of the cyclooxygenase, lipoxygenase, and P450 pathways using indomethacin, baicalein, and 7-ethoxyresorufin. The response to 20-HETE was associated with depolarization and a sustained increase in the intracellular calcium concentration in renal vascular smooth muscle cells. Patch-clamp studies indicated that 20-HETE significantly reduced mean open time, the open-state probability, and the frequency of opening of a 117-pS K+ channel recorded from renal vascular smooth muscle cells in the cell-attached mode. Microsomes prepared from the renal cortex of dogs produced 20-HETE and 20-carboxyarachidonic acid when incubated with [14C]arachidonic acid. These results indicate that 20-HETE is an endogenous constrictor of canine renal arcuate arteries. The vasoconstrictor response to 20-HETE resembles the myogenic activation of these vessels after elevations in transmural pressure and suggests a potential role for this substance in the regulation of renal vascular tone.
Comparison of the number of dihydropyridine receptors with the number of functional L-type calcium channels in embryonic heart.
Authors: Aiba S , Creazzo T L
Published: June 2012

We compared the density of dihydropyridine (DHP) receptor sites with the density of functional L-type calcium channels in ventricular myocytes from chick heart at embryonic day 11. DHP receptors were quantified by using the DHP antagonist (+)-[3H]PN200-110 and by competition binding with the agonist Bay K 8644. The number of agonist and antagonist binding sites per ventricle was similar (250 +/- 15 and 244 +/- 8 fmol, respectively; mean +/- SEM). The mean number of myocytes per ventricle was 8.57 +/- 0.65 x 10(6), as determined by histological methods. From these data, the number of DHP receptors was calculated to be approximately 17,000 per myocyte or 25 to 26 DHP receptors per square micron of cell membrane, based on a mean myocyte membrane capacitance of 6.7 pF and a specific membrane capacitance of 1 microF/cm2. We next determined the number of functional L-type calcium channels by nonstationary fluctuation analysis with whole-cell patch clamp. The mean number of functional L-type channels per cell was 291 +/- 49 and 131 +/- 10 with Ca2+ and Ba2+ as the charge carriers, which yielded a channel density of 0.50 +/- 0.08 and 0.28 +/- 0.02 per square micron of cell membrane, respectively. From these data, the density of DHP receptor binding sites was determined to be from 50 to 100 times the density of functional L-type calcium channels. The function of the "excess" DHP receptors and the determination of whether the proportion of functional channels increases with development require further investigation.
The effect of exchanger inhibitory peptide (XIP) on sodium-calcium exchange current in guinea pig ventricular cells.
Authors: Chin T K , Spitzer K W , Philipson K D , Bridge J H
Published: June 2012

We investigated the effect of exchanger inhibitory peptide (XIP) on Na-Ca exchange current (INa-Ca) in guinea pig ventricular cells. Cells were voltage-clamped with microelectrodes containing 20 mM Na+ and 14.0 mM EGTA ([Ca]i = 100 nM). An outward putative exchange current was stimulated when extracellular Na+ was reduced from 144 mM to zero (Li+ replaced Na+). This outward current showed a significant dependence on extracellular Ca2+. When Na+ removal was delayed for up to 40 minutes (in the absence of extracellular K+ or the presence of 3.0 mM ouabain to block the Na+ pump), outward INa-Ca increased presumably because [Na]i increased. Time-dependent increases of outward current in the absence of K+ could be abolished by reapplication of K+, which presumably reactivates the Na+ pump and reduces intracellular Na+. This effect is blocked in the presence of 3.0 mM ouabain. The dependence of this current on extracellular Ca2+, its dependence on intracellular Na+, and activation by extracellular Na+ reduction, together with its resistance to ouabain all suggest that it is a Na-Ca exchange current. After dialyzing the cell with 10 microM XIP, outward INa-Ca was largely abolished. This indicates that XIP, which is a rather large molecule, can enter the heart cell via the microelectrode in sufficient quantities to inhibit exchange. Inward INa-Ca was blocked secondary to the blockade of outward INa-Ca. L-type Ca2+ current (ICa) was not measurably affected by XIP.(ABSTRACT TRUNCATED AT 250 WORDS)
Inhibition of nitric oxide formation in the nucleus tractus solitarius increases renal sympathetic nerve activity in rabbits.
Authors: Harada S , Tokunaga S , Momohara M , Masaki H , Tagawa T , Imaizumi T , Takeshita A
Published: June 2012

It has been shown that nitric oxide (NO) is synthesized in the central nervous system as well as in vascular endothelial cells. However, the physiological role of NO in cardiovascular regulation by the central nervous system remains unclear. This objective of this study was to examine the possibility that NO plays a role in neural transmission in the nucleus tractus solitarius (NTS) and thus contributes to control of sympathetic nerve activity in rabbits. We examined the effects of NG-monomethyl-L-arginine (L-NMMA), an inhibitor of the formation of NO from L-arginine, microinjected into the NTS on arterial pressure (AP), heart rate (HR), and renal sympathetic nerve activity (RSNA). L-NMMA increased AP and RSNA in rabbits with intact as well as denervated sinoaortic baroreceptors and vagi. L-NMMA increased HR only in rabbits with sinoaortic denervation and vagotomy. Pretreatment with L-arginine microinjected into the NTS, which did not alter baseline AP, HR, and RSNA, prevented the increases in AP and RSNA evoked with subsequent L-NMMA. Pretreatment with D-arginine did not alter the effects of subsequent L-NMMA injections into the NTS. The gain of arterial baroreflex control of RSNA assessed by the slope of the regression line relating changes in AP and those in RSNA caused by intravenous phenylephrine or nitroglycerin did not differ before and after microinjections of L-NMMA. L-NMMA microinjected into the area postrema did not alter AP, HR, or RSNA. These results suggest that in rabbits NO is involved in the mechanism in the NTS that mediates tonic inhibition of RSNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Mechanisms of Ca(2+)-independent contraction in single permeabilized ferret aorta cells.
Authors: Katsuyama H , Morgan K G
Published: June 2012

The mechanisms by which prostaglandin F2 alpha (PGF2 alpha) can cause contractions at constant intracellular Ca2+ were investigated by the direct measurement of force from single saponin-permeabilized smooth muscle cells from the ferret aorta. The size of PGF2 alpha contractions did not change between pCa 9.0 and pCa 6.6. The remainder of the experiments were carried out at pCa 7.0. At pCa 7.0, PGF2 alpha (0.1-100 microM) induced sustained force in a dose-dependent manner, reaching a maximum (2.61 +/- 0.20 microN, n = 14) in 10 minutes. Both protein kinase C pseudosubstrate inhibitor (3 microM) and staurosporine (1 microM) significantly inhibited PGF2 alpha (100 microM)-induced contractions, but staurosporine was more effective. Staurosporine caused 88.8 +/- 13.3% inhibition, whereas protein kinase C pseudosubstrate inhibitor inhibited 62.3 +/- 9.6% of the PGF2 alpha-induced contraction. An inhibitor of type-1 and type-2A protein phosphatases, microcystin-LR, at a concentration of 1 microM induced a gradual and sustained contraction (1.53 +/- 0.21 microN). A lower concentration of microcystin-LR (100 nM) also induced a small but significant contraction (0.36 +/- 0.26 microN). Pretreatment with both 1 microM and 100 nM microcystin-LR caused significant inhibition of the PGF2 alpha-induced contraction from 2.61 +/- 0.20 microN (n = 14) to 0.32 +/- 0.20 microN (n = 6) (p < 0.01) and 1.52 +/- 0.21 microN (n = 6) (p < 0.01), respectively. These results indicate that the part of the PGF2 alpha-induced contraction that occurs at a constant, low intracellular Ca2+ is the combined result of activation of protein kinase C and phosphatase inhibition.
Gene injection into canine myocardium as a useful model for studying gene expression in the heart of large mammals.
Authors: von Harsdorf R , Schott R J , Shen Y T , Vatner S F , Mahdavi V , Nadal-Ginard B
Published: June 2012

We have investigated the regulated expression of genes injected into the heart of large mammals in situ. Reporter constructs using the chloramphenicol acetyltransferase gene under the control of muscle-specific beta-myosin heavy chain (beta-MHC) or promiscuous (mouse sarcoma virus) promoters were injected into the canine myocardium. There was a linear dose-response relation between the level of gene expression and the quantity of plasmid DNA injected between 10 and 200 micrograms per injection site. The level of reporter gene expression did not correlate with the amount of injury imposed on the cardiac tissue. There was no regional variation in expression of injected reporter genes throughout the left ventricular wall. By use of both the mouse sarcoma virus and a muscle-specific beta-MHC promoter, reporter gene expression was one to two orders of magnitude greater in the heart than in skeletal muscle. Expression in the left ventricle was threefold higher than in the right ventricle. Chloramphenicol acetyltransferase activity was detected at 3, 7, 14, and 21 days after injection, with maximal expression at 7 days after injection. Statistical analysis of coinjection experiments revealed that coinjection of a second gene construct (Rous sarcoma virus-luciferase) is useful in the control of transfection efficiency in vivo. Furthermore, using reporter constructs containing serial deletions of the 5' flanking region of the beta-MHC gene, we performed a series of experiments that demonstrate the utility of this model in mapping promoter regions and identifying important regulatory gene sequences in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)
Platelet-activating factor receptor-dependent activation of the muscarinic K+ current in bullfrog atrial myocytes.
Authors: Ramos-Franco J , Lo C F , Breitwieser G E
Published: June 2012

Platelet-activating factor (PAF), a potent signaling lipid implicated as a mediator of pathological responses, has both negative chronotropic and inotropic effects on the heart, although the mechanism(s) involved is not well defined. Because activation of the muscarinic acetylcholine-activated K+ current (IK(ACh)) also produces a negative chronotropic and inotropic response in myocardium, this study examines whether PAF has effects on IK(ACh) in isolated bullfrog atrial myocytes under whole-cell voltage-clamp conditions. We find that 2 microM PAF increases the rate of GTP-gamma-S-mediated IK(ACh) activation (from 0.30 +/- 0.01 min-1 [n = 20] to 0.73 +/- 0.07 min-1 [n = 12], p < 0.005, in the absence of acetylcholine). This effect of 2 microM PAF was blocked by the PAF antagonist CV-3988 (5 microM, 0.33 +/- 0.14 min-1 [n = 12]), suggesting the presence of specific PAF receptors coupled to IK(ACh) activation. Further support for mediation by specific G protein-coupled PAF receptors derives from the inability of PAF to modulate IK(ACh) after maximal activation in the presence of GTP-gamma-S. Eicosatetraynoic acid (ETYA, an inhibitor of 5- and 12-lipoxygenases) did not prevent the PAF-mediated increase in the rate of IK(ACh) activation (10 microM ETYA, 0.28 +/- 0.03 min-1 [n = 7]; 10 microM ETYA plus 2 microM PAF, 0.58 +/- 0.13 min-1 [n = 8]; p < 0.05), suggesting that the observed PAF effect is not mediated by increases in arachidonic acid metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)
Intense sympathetic stimulation releases neuropeptide Y but fails to evoke sustained coronary vasoconstriction in dogs.
Authors: Otani N , Yang T , Levy M N
Published: June 2012

We determined whether a 3-minute period of intense cardiac sympathetic stimulation, which is known to release neuropeptide Y (NPY), elicits a sustained poststimulatory coronary vasoconstriction in anesthetized dogs that had received propranolol. We also periodically measured the cardiac chronotropic responses to test vagal stimulations; these responses served as an index of the neuronal release of NPY. In a group of 11 animals, the coronary vascular resistance increased by 14 +/- 4% during the sympathetic stimulation. After cessation of stimulation, however, coronary vascular resistance returned rapidly to its control value. The cardiac responses to the test vagal stimuli were attenuated by approximately 40% after cessation of sympathetic stimulation, and this inhibitory effect persisted for approximately 60 minutes. In a second group of eight dogs, we determined whether the intense sympathetic stimulation potentiates the coronary vascular responses to exogenous norepinephrine (NE). Before sympathetic stimulation, standard intracoronary infusions of NE increased coronary vascular resistance by 14 +/- 2%. Intense antecedent sympathetic stimulation did not alter the coronary vascular responses to subsequent NE infusions. However, the chronotropic responses to test vagal stimuli were initially attenuated by approximately 30%, and this inhibitory effect persisted for approximately 1 hour. In a third group of four dogs, we found that exogenous NPY significantly potentiated the coronary vasoconstriction evoked by NE infusions. The coronary vascular responses to combined infusions of NE and NPY were consistently greater (by approximately 13%) than the sum of the responses to these substances when they were infused separately. We conclude that, even though sufficient NPY appears to be released from the sympathetic nerve endings to inhibit vagal neurotransmission, the quantity of NPY released into the coronary blood vessels under the conditions of our experiments appears to be insufficient either to elicit a sustained coronary vasoconstriction or to potentiate the vasoconstrictor effects of intracoronary NE infusions.
Single delayed rectifier channels in the membrane of rabbit ventricular myocytes.
Authors: Veldkamp M W , van Ginneken A C , Bouman L N
Published: June 2012

In rabbit ventricular cells, the delayed rectifier current (IK) has not been extensively studied, and properties of single IK channels still need to be determined. In this study, we present data on a voltage-dependent channel in rabbit ventricular cells; the properties indicate that it is an IK channel. Patch-clamp experiments were carried out on cell-attached and inside-out patches of rabbit ventricular cells. Single-channel currents were recorded at negative potentials as inward currents with 150 mM K+ in the pipette. Voltage-dependent channel activity was only present after the return from a depolarizing test pulse, indicating activation on depolarization. Single-channel conductance calculated from the current-voltage relation was 13.1 pS (pooled data, n = 8). The shift in reversal potential of the unitary currents, determined at 150 and 300 mM K+ at the intracellular side of the membrane, showed that the channels were highly permeable to potassium ions. Increase of the duration or the amplitude of the depolarizing test pulse increased channel activity. The time constant for activation at +30 mV was 187 msec (pooled data, n = 4). Half-activation potential was -4.9 +/- 3.8 mV (mean +/- SD), and the slope factor was 7.2 +/- 3.7 mV (mean +/- SD). Current tails, reconstructed from averaged single-channel currents, revealed that the time course of deactivation decreased from 694 +/- 73 msec at -80 mV to 136 +/- 39 msec at -110 mV. Additional evidence that the channel was indeed an IK channel was provided by the observation that the channel was blocked by 10(-7) M E-4031, a class III antiarrhythmic agent that has been shown to block a component of the macroscopic IK in guinea pig heart.
In vivo observation of subendocardial microvessels of the beating porcine heart using a needle-probe videomicroscope with a CCD camera.
Authors: Yada T , Hiramatsu O , Kimura A , Goto M , Ogasawara Y , Tsujioka K , Yamamori S , Ohno K , Hosaka H , Kajiya F
Published: June 2012

We developed a portable needle-probe videomicroscope with a charge-coupled device (CCD) camera to visualize the subendocardial microcirculation. In 12 open-chest anesthetized pigs, the sheathed needle probe with a doughnut-shaped balloon and a microtube for flushing away the intervening blood was introduced into the left ventricle through an incision in the left atrial appendage via the mitral valve. Images of the subendocardial microcirculation of the beating heart magnified by 200 or 400 on a 15-in. monitor were obtained. The phasic diameter change in subendocardial arterioles during cardiac cycle was from 114 +/- 46 microns (mean +/- SD) in end diastole to 84 +/- 26 microns in end systole (p < 0.001, n = 13, ratio of change = 24%) and that in venules from 134 +/- 60 microns to 109 +/- 45 microns (p < 0.001, n = 15, ratio of change = 17%). In contrast, the diameter of subepicardial arterioles was almost unchanged (2% decrease, n = 5, p < 0.01), and the venular diameter increased by 19% (n = 8, p < 0.001) from end diastole to end systole. Partial kinking and/or pinching of vessels was observed in some segments of subendocardial arterioles and venules. The percentage of systolic decrease in the diameter from diastole in the larger (> 100 microns) subendocardial arterioles and venules was greater than smaller (50-100 microns) vessels (both p < 0.05). In conclusion, using a newly developed microscope system, we were able to observe the subendocardial vessels in diastole and systole.(ABSTRACT TRUNCATED AT 250 WORDS)
Oral contraceptive treatment decreases arterial low density lipoprotein degradation in female cynomolgus monkeys.
Authors: Wagner J D , Adams M R , Schwenke D C , Clarkson T B
Published: June 2012

The effect of oral contraceptive therapy on early events in atherogenesis was studied in female cynomolgus monkeys. After a 1-month dietary challenge, monkeys were randomized into three groups stratified by total plasma cholesterol and high density lipoprotein cholesterol concentrations. The monkeys were then fed a cholesterol-containing diet for 16 weeks. This relatively short period ensured that studies were done before any treatment-induced differences in arterial morphology occurred. Monkeys were treated with either diet alone (control group), with the addition of a monophasic oral contraceptive (equivalent to a human dose of 50 micrograms ethinyl estradiol and 500 micrograms norgestrel per day), or with a triphasic oral contraceptive (equivalent to a human dose of 30-40 micrograms ethinyl estradiol and 50-125 micrograms levonorgestrel per day). Twenty-four hours before necropsy, low density lipoproteins (LDLs) labeled with 131I and LDLs labeled with the residualizing label 125I-tyramine cellobiose were injected into the animals. The arterial LDL degradation rate, amount of undegraded LDLs, and total LDL accumulation were then determined. Although there were regional differences in LDL metabolism, both treatments decreased the rate of LDL degradation and LDL accumulation in the coronary arteries and other arterial sites. Treatment also resulted in significantly lower LDL molecular weights. Despite a trend toward a more atherogenic lipid profile (decreased high density lipoprotein cholesterol and increased total plasma/high density lipoprotein cholesterol ratio), oral contraceptive treatment may inhibit atherogenesis by decreasing arterial LDL degradation.
Molecular cloning of three isoforms of the L-type voltage-dependent calcium channel beta subunit from normal human heart.
Authors: Collin T , Wang J J , Nargeot J , Schwartz A
Published: June 2012

We have cloned and sequenced cDNAs for three isoforms of the L-type voltage-dependent calcium channel beta subunit isolated from a normal human heart cDNA library. One of these subunits, designated beta a, hybridized with a 3.4-kb message, and the other two, designated beta b and beta c, hybridized with a message of approximately 1.9 kb. The presence of both of these latter two messages in human heart was confirmed by polymerase chain reaction methodology. Considering the differences between beta a and beta b/beta c, we find it likely that these messages may be encoded by two different gene sequences, beta a and beta b/beta c, and that the beta b/beta c sequence can be alternatively spliced in the 209-260 region. The data suggest that the human heart presents a different pattern of beta subunit expression from that found in the rat and rabbit heart.
Low density lipoprotein subfractions and [Ca2+]i in vascular smooth muscle cells.
Authors: Weisser B , Locher R , de Graaf J , Vetter W
Published: June 2012

Several studies have established that plasma low density lipoprotein (LDL) consists of various discrete subfractions. Using a variety of techniques (analytical ultracentrifugation, equilibrium density gradient ultracentrifugation, and gradient gel electrophoresis), LDL has been fractionated into a maximum of seven subclasses that differ in particle size, density, and physiochemical composition. Recently, a predominance of smaller denser LDL particles has been associated with an increased risk of coronary artery disease. However, other lipoprotein changes, such as elevated triglycerides and lower HDL cholesterol levels, have been shown in patients with a predominance of the smaller denser LDL subfractions. Thus, it is unclear whether the enhanced atherogenic potential is induced by the LDL subfraction pattern per se or by concomitant lipoprotein changes. Because intracellular free Ca2+ is an important second messenger involved in atherogenesis and regulation of vascular tone, we studied the influence of three LDL subfractions (very light [LDL1], 1.030 to 1.033 g/mL; light [LDL2], 1.033 to 1.040 g/mL; and dense [LDL3], 1.040 to 1.045 g/mL) on [Ca2+]i in vascular smooth muscle cells (VSMCs) cultured from rat aorta. LDL subfractions were isolated by density gradient ultracentrifugation from human EDTA-plasma (n = 15). [Ca2+]i was measured by fura 2 fluorescence. Basal [Ca2+]i was 77 +/- 6 nmol/L. Stimulation of VSMCs with dense LDL3 caused a significantly (P < .05) more pronounced increase (+71 +/- 13 nmol/L) compared with LDL1 (+38 +/- 8 nmol/L) and LDL2 (+36 +/- 9 nmol/L). To further investigate the mechanisms leading to the stimulation of [Ca2+]i by LDL subfractions, we incubated VSMCs with the Ca2+ antagonists nifedipine, diltiazem, and verapamil in concentrations up to 10 mumol/L.(ABSTRACT TRUNCATED AT 250 WORDS)
Regulation of capillary perfusion by small arterioles is spatially organized.
Authors: Frame M D , Sarelius I H
Published: June 2012

To explore a mechanism for spatial recruitment of capillaries, this study determined whether the arterioles controlling capillary perfusion, which typically arise as sequential branches along a transverse arteriole, could respond differently from each other in situ in a spatially ordered way. Diameter changes were measured for these arterioles at a known location in the intact microvasculature in the cremaster muscle of anesthetized Golden hamsters (N = 67); each arteriole controls separate capillary groups. These arterioles all had the same concentration dependence to locally (by micropipette) applied norepinephrine (NE, 10(-9) to 10(-3) mol/L), and 10(-9) mol/L NE did not induce diameter changes when applied locally to individual vessels. However, 10(-9) mol/L NE added to the tissue superfusate, or 5% added superfusate oxygen (also locally subthreshold), each induced significant diameter changes (both constrictions and dilations), in different branches, that were presumably due to summation of individually subthreshold events that changed the prevailing conditions at the point of observation. These significant diameter changes were related to the maximal diameter or to initial tone of the branches, but these changes occurred in different ways for NE versus oxygen. With NE, the branch arterioles that constricted (versus dilated) were significantly larger (maximal diameter, 22.3 +/- 2.6 versus 15.9 +/- 2.1 microns) and had higher tone (fractional constriction, 0.53 +/- 0.05 versus 0.63 +/- 0.05); with oxygen, those that constricted were the same size as those that dilated (maximal diameter, 28.6 +/- 1.1 versus 30.5 +/- 2.7 microns), but constrictors had lower tone (fractional constriction, 0.49 +/- 0.04 versus 0.39 +/- 0.06).(ABSTRACT TRUNCATED AT 250 WORDS)
Nitric oxide formation contributes to beta-adrenergic dilation of resistance coronary vessels in conscious dogs.
Authors: Parent R , al-Obaidi M , Lavallée M
Published: June 2012

The contribution of the L-arginine/nitric oxide pathway to beta-adrenergic dilation of resistance coronary vessels was examined in conscious dogs instrumented for measuring coronary blood flow (CBF), left ventricular (LV) wall thickening, and LV and aortic pressures and for intracoronary injections of acetylcholine (0.003 micrograms/kg), nitroglycerin (0.175 micrograms/kg), and graded doses of isoproterenol (0.0005 to 0.004 micrograms/kg). Peak increases in CBF with intracoronary isoproterenol (0.001 micrograms/kg) averaged 105 +/- 10% from baseline. With acetylcholine, CBF increased by 158 +/- 11%, and with nitroglycerin, CBF increased by 139 +/- 10%. After the administration of intracoronary N omega-nitro-L-arginine methyl ester (L-NAME, 10 micrograms/kg per minute for 12 minutes) to block nitric oxide synthesis from L-arginine, baseline CBF was not altered, and CBF increased by 49 +/- 7% with isoproterenol and by 94 +/- 6% with acetylcholine; both values were smaller (P < .01) than those before the arginine analogue. With nitroglycerin, CBF was increased by 145 +/- 11%, not significantly different from the value before L-NAME. Intracoronary L-arginine (1.0 mg/kg per minute for 12 minutes), the precursor of nitric oxide synthesis, partially reversed the inhibition of L-NAME on CBF responses to acetylcholine and isoproterenol. After beta 1-adrenergic blockade, CBF responses to isoproterenol and acetylcholine were also reduced (P < .05) by the arginine analogue. When increases in CBF were prevented, peak changes in coronary vascular conductance with intracoronary bolus doses of acetylcholine and isoproterenol were attenuated (P < .01) by L-NAME. Thus, nitric oxide formation is an important intermediate in beta-adrenergic dilation of resistance coronary vessels in conscious dogs.
Na(+)-H+ exchange inhibitors decrease neointimal formation after rat carotid injury. Effects on smooth muscle cell migration and proliferation.
Authors: Mitsuka M , Nagae M , Berk B C
Published: June 2012

The presence of multiple growth stimuli at the sites of vascular injury following angioplasty suggests that therapies targeted toward common growth pathways will be more effective than therapies that inhibit only a single growth factor. We tested this hypothesis using amiloride and ethyl isopropyl amiloride (EIPA), which are inhibitors of the Na(+)-H+ exchanger, whose activity is required in many cells for proliferation and migration. In the rat carotid injury model, EIPA (100 micrograms/h for 15 days) significantly decreased intimal area and the ratio of intimal to medial area, whereas amiloride (25 micrograms/h) showed an inhibitory trend that was similar to that observed for captopril (80 mg/kg per day) and heparin (25 U/h). EIPA and amiloride inhibited rat vascular smooth muscle cell DNA synthesis, with IC50 values of 8.8 and 82.2 microM, respectively. Using platelet-derived growth factor as a chemoattractant, EIPA caused a concentration-dependent inhibition of migration (IC50, approximately 60 microM). Because amiloride and EIPA have nonspecific effects on cellular function (especially inhibition of tyrosine kinases), we sought to characterize the specific role of the Na(+)-H+ exchanger in vascular smooth muscle cell proliferation and migration. We generated a Na(+)-H+ exchanger-deficient mutant cell line [RNHE(-)]. Studies with these cells suggested that the inhibitory effects of EIPA and amiloride were mediated only in part via Na(+)-H+ exchange because (1) RNHE(-) cells grew well at pH 6.8 to 7.5 in bicarbonate-containing medium, and (2) there was no difference in migration in response to platelet-derived growth factor in the RHNE(-) cells. In summary, these data indicate that amiloride and EIPA inhibit neointimal formation in the rat carotid after injury. However, the mechanism of inhibition is likely to involve cellular events other than Na(+)-H+ exchange, such as an effect on tyrosine kinases.