摘要:

In heart cells, several plasma membrane ion channels are targets for phosphorylation. However, it is not known whether sarcoplasmic reticulum (SR) ion channels, which are also essential in the regulation of cardiac function, are regulated by second-messenger systems. Here, we show that a Cl−channel in the cardiac SR membrane is activated by the catalytic subunit of protein kinase A (PKA). Purified cardiac heavy SR vesicles were incorporated into planar lipid bilayers. This channel spontaneously inactivated within a few minutes after the channel was incorporated into the bilayer. Mg-ATP (2–5 mM), but not the nonhydrolyzable ATP analogue 5'-adenylylimidodiphosphate, added to the cis solution prevented this spontaneous channel inactivation. After the inactivation process occurred, the catalytic subunit of PKA (with 0.05 mM Mg-ATP) reactivated this channel. These effects of Mg-ATP and PKA on the Cl−channel were prevented by an inhibitor of PKA. Thus, these results suggest that this SR Cl−channel is a novel target of PKA-dependent phosphorylation in cardiac muscle regulation.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

Regulation of blood flow and mitochondrial respiration in the heart would be clarified by improved knowledge of interstitial concentrations and cellular production rates of adenosine; however, these variables cannot be measured directly. To interpret indexes that are available, a comprehensive mathematical model was developed, based on a large body of experimental data. The model describes most of the important pathways of capillary-tissue transport and cellular metabolism of adenosine in the guinea pig heart. It includes capillary flow, solute transport between tissue regions, nonlinear enzyme kinetics for adenosine kinase and adenosine deaminase, and reversible biunireactant kinetics forS-adenosylhomocysteine hydrolase in cardiomyocytes and endothelial cells, intracellular production of adenosine via AMP hydrolysis and transmethylation, and extracellular production of adenosine. A single set of parameter values for the model was obtained in the first stage of the analysis by taking certain values directly from published sources, other values were subject to specific constraints, and other values were determined by parameter optimization. The effects of flow and endothelial metabolism on the relation between interstitial and venous adenosine concentrations were determined. The relation between myocardial adenosine production rate andS-adenosylhomocysteine accumulation in the presence of excess homocysteine was estimated. In the second stage of the analysis, the model was used to investigate the mechanism of myocardial adenosine production, without changing the parameter values. Cellular adenosine production rates were estimated by fitting measurements of venous adenosine release obtained during altered energetic conditions in experiments by different investigators. The original results showed a dissociation between measurements of cytosolic AMP concentrations and venous adenosine release. It is concluded that 1) it is essential to account for the effect of flow on interstitial and venous adenosine concentrations, since decreased flow may produce effects outwardly resembling inhibition of the enzyme 5'-nucleotidase, 2) adenosine concentrations in epicardial transudate are not in equilibrium with interstitial fluid, and 3) the rate of cellular adenosine production increases monotonically with free cytosolic concentrations of AMP during a variety of alterations in energy balance of the guinea pig heart.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

Mitochondrial calcium overload has been suggested as a marker for irreversible injury in the ischemic heart. A new technique is used to measure dynamic changes in mitochondrial free calcium concentration ([Ca2+]m) in electrically stimulated (0.2 Hz) adult rat cardiac myocytes during exposure to anoxia and reoxygenation. Cells were incubated with indo-1 AM, which distributes in both the cytosol and mitochondria. After Mn2+quenching of the cytosolic signal, cells were exposed to anoxia, and the residual fluorescence was monitored. [Ca2+]. averaged 94±3 nM (n=16) at baseline, less than the baseline diastolic cytosolic free calcium concentration ([Ca2+]c, 124±4 nM,n=12), which was measured in cells loaded with the pentapotassium salt of indo-1. [Ca2+]mand [Ca2+]crose steadily only after the onset of ATP-depletion rigor contracture. At reoxygenation 35 minutes later, [Ca2+]cfell rapidly to preanoxic levels and then often showed a transient further rise. In contrast, [Ca2+]mshowed only a slight transient fall and a secondary rise at reoxygenation. At reoxygenation, cells immediately either recovered, demonstrating partial relengthening and retaining their rectangular shape and response to stimulation, or they hypercontracted to rounded dysfunctional forms. Recovery occurred only in cells in which [Ca2+]mor [Ca2+]cremained below 250 nM before reoxygenation. Early during reoxygenation, [Ca2+]mremained higher in cells that hypercontracted (305±36 nM) than in cells that recovered (138±9 nM,p<0.05), whereas [Ca2+]cdid not differ between the two groups (156±10 versus 128±10 nM, respectively; p=NS). The role of the sarcoplasmic reticulum in Ca2+regulation was evaluated in cells (n=16) pretreated with thapsigargin, an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase. During anoxia [Ca2+]cand [Ca2+]mrose as they did without thapsigargin pretreatment. At reoxygenation, the rapid fall in [Ca2+]cwas blunted, and [Ca2+]mshowed an immediate increase in these cells, demonstrating the importance of the sarcoplasmic reticulum in postanoxic Ca2+regulation. In summary, cellular hypercontracture is not associated with a sudden and massive rise in [Ca2+]cimmediately after reoxygenation. The basis for the relation between [Ca2+]mand cellular recovery as well as the mechanisms underlying the observed changes in [Ca2+]mremain to be defined.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

Oxidatively modified low density lipoprotein (oxidized LDL), an atherogenic lipoprotein that exists in the atherosclerotic arteries, has been shown to alter endothelial cell functions. In the present study, we examined the effects of oxidized LDL on the production of endothelin-1-like immunoreactivity (ET-1-LI) by the cultured vascular endothelial cells from porcine aorta and human umbilical vein. Incubation with oxidized LDL resulted in a dose-dependent suppression of ET-1-LI release by both endothelial cells. Oxidized LDL also inhibited thrombin-mediated stimulation of ET-1-LI secretion. However, native LDL had no effects on ET-1-LI secretion. A lipid extract from oxidized LDL, but not from native LDL, inhibited ET-1-LI secretion, indicating that the lipid component of oxidized LDL was required for the inhibition of ET-1-LI secretion. Oxidative modification of LDL was associated with degradation of a substantial amount of phosphatidylcholine to lysophosphatidylcholine (LPC). Pretreatment with defatted albumin, which is an acceptor for hydrophilic lipids including LPC, reduced LPC concentration in oxidized LDL to that in native LDL and simultaneously prevented the inhibitory effects of oxidized LDL on ET-1-LI secretion. Incubation with synthetic LPC (palmitoyl), but not with synthetic phosphatidylcholine (dipalmitoyl), suppressed ET-1-LI secretion by the endothelial cells. No cell death was observed during the incubations as judged by the trypan blue exclusion test, and protein synthesis of the endothelial cells was not affected by lipids or lipoproteins at a concentration at which suppression of ET-1-LI was observed. We concluded that LPC in oxidized LDL causes suppression of ET-1-LI release, which may counteract the vasoconstrictive properties of atherosclerotic arteries.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

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 HRS/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.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

Endothelin may play an important role in modulating myocardial contractility under certain pathophysiological conditions. To determine whether endothelin beneficially modulates myocardial contractility in the common clinical condition of acidosis, we compared the effects of endothelin-1 on intracellular Ca2+transients and isometric contractions under normal (extracellular pH [pHo] 7.4) and acidotic (pHo6.4) conditions in ferret papillary muscles (n=33) loaded with the Ca2+-regulated bioluminescent indicator aequorin. A pHoof 6.4 was induced by replacing 92% of HCO3−with Cl−in the bathing medium. The effects of endothelin at pHo6.4 differed from the effects at pHo7.4 in that 1) the minimally effective concentration of endothelin was 30-fold lower (1 × 10−10M at pHo6.4; 3 × 10−9M at pHo7.4) and the concentration-response curve of endothelin was significantly shifted to the left with a decrease in log EC50from −7.83±0.13 to −8.92±0.10 (p<0.001), indicating an increased sensitivity of myocardium to endothelin; 2) endothelin produced an increase of ≈375% in tension development at pHo6.4 (≈62% at pHo7.4) (p<0.001) without increasing peak [Ca2+]i(≈13% increase at pHo7.4,p<0.001), indicating an increase in myofilament Ca2+responsiveness; and 3) endothelin significantly abbreviated (≈ −19%,p<0.001) the prolonged intracellular Ca2+transient induced by acidosis (pHo6.4). In addition, pretreatment with 10 μM of the Na+-H+exchange inhibitor 5-(N-methyl-N-isobutyl)-amiloride significantly attenuated endothelin-induced effects on the intracellular Ca2+transient and contraction during acidosis. Results indicate that 1) acidosis decreased myofilament Ca2+responsiveness and prolonged the intracellular Ca2+transient, whereas endothelin enhanced myofilament Ca2+responsiveness and abbreviated the intracellular Ca2+transient by decreasing intracellular H+via Na+-H+exchange during acidosis; and 2) endothelin exerts its cardiotonic effects at much lower concentrations during acidosis, presumably due to altered behavior of the receptor. Taken together, these findings suggest that endothelin could beneficially reverse acidosis-induced negative inotropic and lusitropic effects on the intracellular Ca2+transient and myocardial contractility.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

The dominant mechanism responsible for restenosis after angioplasty is believed to be the activation of medial smooth muscle cells (SMCs), leading to their proliferation, migration to the subintima, and further proliferation. To develop novel strategies that might inhibit or prevent restenosis, we previously used a chimeric toxin composed of transforming growth factor-α (which targets the epidermal growth factor receptor) and mutatedPseudomonasexotoxin to preferentially recognize and kill rapidly proliferating, versus quiescent, vascular SMCs. We have recently cloned and expressed a recombinant gene encodingPseudomonasexotoxin with a mutated (nonfunctional) cell recognition domain fused with the ligand acidic fibroblast growth factor, termed aFGF-PE664GluKDEL; thus, this recombinant toxin targets the fibroblast growth factor receptor. In the present stud, we evaluated the relative effects of this chimeric toxin on quiescent versus rapidly proliferating vascular SMCs and also determined whether aFGF-PE664GluUKDEL exerted different effects on SMCs versus endothelial cells. Rapidly proliferating SMCs (grown in 10% fetal bovine serum) were very sensitive to the cytotoxic effects of aFGF-PE664GluKDEL, whereas cytotoxicity was significantly less when the SMCs were in a quiescent state (grown in medium supplemented with 0.5% fetal bovine serum). The chimeric toxin was also significantly less cytotoxic against endothelial cells. Competition studies using excess acidic fibroblast growth factor indicated that the cytotoxic effects are specifically mediated by the fibroblast growth factor receptor. Thus, the present studies suggest a potentially expanded role of recombinant toxin therapy in restenosis: multiple receptors can be targeted, and cytotoxic effects, at least in vitro, can be preferentially directed to rapidly proliferating vascular SMCs, with relative sparing of vascular endothelial cells. It will next be necessary to test this strategy for inhibiting restenosis in an in vivo model of vascular injury and SMC proliferation.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

Platelet-derived growth factor (PDGF) is believed to be a critical mediator of vascular smooth muscle cell (SMC) proliferation. Because insulin-like growth factor (IGF) I (IGF-I) functions as a progression factor for the mitogenic effects of PDGF, we hypothesized that IGF-I gene expression and the production of IGF binding proteins (IGFBPs) by cultured rat aortic SMCs might be regulated by one or more of the three isoforms of PDGF: PDGF-AA, -BB, and -AB. IGF-I gene expression was highly dependent on cell density: IGF-I mRNA transcripts decreased markedly as a function of cell confluence. IGF-I mRNA content was inhibited to a similar degree by PDGF-AA, -BB, and -AB through a mechanism requiring protein synthesis. The inhibition was readily apparent at 4 hours, reaching approximately 25% of control levels after 24 hours. Radioimmunoassayable IGF-I was only barely detectable in SMC-conditioned serum-free medium and not significantly modulated by PDGF. Western ligand blot revealed that vascular SMCs release 30-kd and 24-kd IGFBP into serum-free conditioned medium. PDGF isoforms did not significantly alter release of the 30-kd IGFBP but evoked a fivefold to sixfold increase in the 24-kd IGFBP. The 24-kd IGFBP was found to comigrate with IGFBP-4, a recently identified binding protein that inhibits IGF action. The 30-kd protein was not merely a glycosylated form of IGFBP-4, because it was not sensitive to N-glycanase digestion. PDGF-AA, -BB, and -AB markedly induced expression of IGFBP-4 mRNA in a time- and concentration-dependent fashion. Vascular SMCs also express IGFBP-2 mRNA, but its abundance was not induced by PDGF. In conclusion, PDGF evokes a complex pattern of regulation of genes in the IGF/IGFBP system. By inhibiting IGF-I production and specifically inducing biosynthesis of the inhibitory binding protein IGFBP-4, PDGF may set in motion mechanisms to limit the final magnitude of the mitogenic response.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

We carried out a systematic study on the effects of elevating [K]Oon the properties of a transient outward potassium channel encoded by a cardiac cDNA (RHKI) and compared them with those on twoShakerpotassium channels (H-4 and H-37). The amino acid sequences of all three channels are known, and their structure-function relations have been partially characterized. All three channels were expressed inXenopusoocytes and studied under double-microelectrode voltage-clamp conditions. For all three channels, elevating [K]ocaused an increase in the channels' chord conductances and a negative shift in the calculated activation curves. However, in other aspects of channel properties that are related to the channels' inactivation processes, there were differences in the changes induced by increasing [K].: 1) Elevating [K]. caused a positive shift in the steady-state inactivation curves of RHK1 and H-4 but did not cause any shift in H-37.2) Elevating [K]. slowed the time course of inactivation of H-37 but did not cause any significant changes in the time course of RHK1 or H-4. 3) Elevating [K]0accelerated the rate of recovery from inactivation of RHK1 and H-4 but slowed the recovery time course of H-37. Our experiments show that elevating [K]0can cause a wide range of effects on the transient outward potassium channels. Furthermore, raising [K]0induced similar changes in RHK1 and H-4 (inactivation mediated by an “N-type” mechanism) that were different from the changes in H-37 (inactivation mediated by a “C-type” mechanism). Therefore, our data suggest that part of the effects of elevating [K]0on channel properties may depend on the channel's inactivation mechanism. This hypothesis is supported by results from experiments studying the effects of elevating [K]0on a mutant RHK1 channel (RHK1A3-25), which apparently lacks the N-type and C-type inactivation mechanisms.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

α1-Adrenoceptor activation can enhance myocardial contractility, and two possible inotropic mechanisms are an increase in myofilament Ca2+sensitivity and action potential prolongation, which can increase net Ca2+entry into cells. In adult rat ventricular myocytes (bath Ca2+, 1 mM; stimulated at 0.2–0.5 Hz), the drug 4-aminopyridine and the whole-cell voltage clamp have been used to control Ca2+entry and differentiate between the two mechanisms. At 22–23°C the specific α1-adrenoceptor agonist methoxamine (100 μM) prolonged action potential duration at 50% repolarization from 55±2 to 81±5 msec, delayed time to peak contraction, and increased shortening amplitude from 5.3±0.6 to 7.8±1 μm (n=18). Reduction of the transient outward current and other K+currents by methoxamine was the major cause of action potential prolongation in rat myocytes with little change in the L-type calcium current. Block of the transient outward current with 2 mM 4-aminopyridine prolonged action potential duration from 52±6 to 98±12 msec and increased unloaded cell shortening from 2.9±0.4 to 6.6±0.6 p.m (n=4). Subsequently, methoxamine no longer increased cell shortening, although significant potentiation of twitch amplitude was still seen after a brief rest interval. In voltage-clamp experiments, with 70–500-msec pulses, although membrane currents were reduced, methoxamine had no positive inotropic effect and reduced cell shortening from 5.3±0.7 to 4.97±0.8 μm at pulse potentials positive to −40 mV. Similar al-adrenoceptor responses were observed at 35°C during action potential and voltage-clamp experiments, which could be blocked by 10 μM prazosin. In myocytes loaded with the Ca2+indicator indo-1, α1-adrenoceptor stimulation or 4-aminopyridine both increased cell contraction and intracellular Ca2+transients by similar amounts. As in unloaded cells, prior exposure to 4-aminopyridine prevented any inotropic effect of methoxamine without changing the systolic intracellular Ca2+transient. The results indicated that under our experimental conditions positive inotropy in rat cardiomyocytes on exposure to α1-adrenoceptor agonists was strongly correlated with the action potential prolongation that accompanied K+current reduction. In addition, modulation of K+channels could occur independent of changes in contractility and/or [Ca2+];.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID