摘要:

Fatty acid influx into human myocardium was studied in 15 patients during the cooling phase of cardiopulmonary by pass at myocardial temperatures of 37° to 25°C. The fitting of the data to a functional relation, developed in this study, revealed fatty acid influx to be a temperature-dependent saturable process corresponding to a Michaelis-Menten constant(Km)at 37°C of 0.26 ± 0.084, μmol/g, a maximal fatty acid influx velocity (Kmax.) at 37°C of 0.28 ± 0.045 μmol/g per minute, activation energy for fatty acid binding to the putative carrier (E) of 23.8±5.6 kcal/mol, and a free energy for conformational change of the carrier (U) of 10.9 ± 8.0 kcal/mol. In short-term cultured hepatocytes, Kmincreased in the absence of Na+from 171 ± 48 to 301±71 nmol/L, and Vmaxof [3H] oleate decreased from 1063 ± 69 to 847 ± 68 pmol/min per milligram protein. The fitting of these data to a functional relation revealed a transmembrane potential-dependent component of parameters E and U to be −0.479 and −0.374 kcal/mol, respectively. It is proposed that for fatty acid influx a protonated fatty acid form is preferred that consists of a Na+complex with the mesomeric form of nondissociated fatty acid from which Na+and H+are released during collision with the carrier.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

Polytetrafluoroethylene (PTFE) grafts placed into the arterial circulation of baboons for 8 weeks under high blood flow (HF) conditions develop a thin intima composed of smooth muscle cells (SMCs) and extracellular matrix beneath an endothelial monolayer. When these grafts are returned abruptly to normal flow (NF), they develop marked intimal thickening within 1 month. The mechanisms underlying this thickening are unclear. We studied the SMC response to altered flow by placing bilateral aortoiliac PTFE grafts into baboons with bilateral femoral arteriovenous fistulas. After 8 weeks, one fistula was closed, returning the graft flow on that side to NF. The opposite graft remained under HF conditions. Flow differences were monitored with duplex ultrasound (for all grafts: NF, 135±21 [mean±SEM] mL/min; HF, 507±35 mL/min; P<.001). Grafts were removed 2, 4, 7, 14, or 28 days later (five animals per group). Endothelial coverage, as assessed by scanning electron microscopy, was intact in each graft. Intimal area and SMC number increased progressively in NF grafts through 28 days (for area: NF, 3.0±0.3 mm2; P<.001; and for SMCsper cross section: NF, 11.8±1.1x103; HF, 2.6±1.Ox103; P<.002). Intimal SMC proliferation (thymidine labeling) was increased significantly in NF grafts at 4 and 7 days (at 4 days: NF, 5.9±1.5%; HF, 1.4±0.6%; P<.05). Extracellular matrix accounted for an equal proportion of intimal mass in NF and HF grafts (percent matrix at 28 days: NF, 62.9±1.6%; HF, 63.7±4.7%; P=NS). We conclude that intimal thickening in this model of flow-induced vascular remodeling is due to increased SMC proliferation and accumulation of SMCswith a proportionate amount of extracellular matrix.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

Experiments were designed to examine whether or not insulin-like growth factor I (IGF-I), which is produced by vascular cells in response to injury, affects the production of nitric oxide evoked by the inducible nitric oxide synthase in cultures of smooth muscle cells from the rat aorta. Nitric oxide production was assessed indirectly by the measurement of nitrite accumulation and nitric oxide synthase activity by determining the formation of L–citrulline from L–arginine. Nitric oxide synthase was induced in vascular smooth muscle cells that had been exposed to interleukin-1β (IL-1β) or tumor necrosis factor-α (TNF-α). IGF-I inhibited, in a concentration-dependent manner, the production of nitrite and L–citrulline evoked by IL-1β or TNF-α. The inhibition caused by IGF-I required the presence of the growth factor during the induction of nitric oxide synthase. Two IGF-I-related proteins, IGF-II and insulin, also inhibited, but to a smaller extent, the release of nitrite and the formation of L–citrulline stimulated by IL-1β. Under bioassay conditions, the perfusates from columns containing IL-1β-treated smooth muscle cells relaxed rings of rat aorta without endothelium that had been contracted with phenyl ephrine; these relaxations were reversed by nitro-L–arginine. Addition of IL-1β-treated vascular smooth muscle cells to indomethacin-treated platelets inhibited their aggregation to thrombin; methylene blue prevented this inhibition. Control smooth muscle cells or cells exposed to IGF-I alone did not have such effects. Smooth muscle cells that had been exposed simultaneously to IL-1β and IGF-I also relaxed detector rat aortic rings, but to a smaller extent, and minimally affected the aggregation. IL-1β caused the expression of inducible nitric oxide synthase mRNA levels in vascular smooth muscle cells; this response was reduced in cells treated with IL-1β in combination with IGF-I. These observations indicate that IGF-I inhibits the cytokine-induced production of nitric oxide by preventing the induction of nitric oxide synthase. They further suggest that IGF-I may be an important modulator of the production of nitric oxide at sites of vascular injury.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

The intracellular pathways for basal atrial natriuretic factor (ANF) secretion from the heart and their correlation with ANF processing to the active form were characterized in cultured neonatal rat atrial and ventricular myocytes. Brefeldin A, a fungal antimetabolite that blocks transport of newly synthesized proteins from the endoplasmic reticulum, was used to inhibit nascent protein trafficking. Thus, release of newly synthesized hormone was blocked, but release of stored hormone was unaffected. Whereas brefeldin A inhibited basal ventricular ANF release to 10% of the control value, basal ANF release from atrial cells was enhanced. Furthermore, basal atrial ANF secretion was inhibited by agents preventing myocyte depolarization, Ca2+influx, release of Ca2+from intracellular stores, or activation of protein kinase C, whereas ventricular ANF secretion was unaffected by these agents. Brefeldin A did not alter maturational processing of pro-ANF to ANF-(99–126) in either atrial or ventricular cultures. These findings indicate that (1) basal secretion of ANF from ventricular cells relies largely on newly synthesized hormone and is probably constitutive, (2) basal secretion of ANF from atrial cells is independent of transport of newly synthesized protein and occurs via a regulated pathway controlled at least in part by signaling changes associated with myocyte beating, and (3) processing of pro-ANF occurs either with constitutive or regulated secretion of hormone, which may indicate multiple cellular locations for the processing enzyme.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

In the rabbit heart, multiple isoforms of cardiac troponin T (cTnT1through cTnT5, from largest in size to smallest), a protein essential for calcium-regulated myofibrillar ATPase activity, have been identified, and a correlation has been found between these isoforms and myofilament sensitivity to calcium. We have sought to establish the molecular basis of this diversity. Restriction-digest analysis of genomic DNA has indicated that the rabbit cTnT gene is a single-copy gene. cTnT cDNA clones were isolated from cDNA libraries, yielding a consensus sequence for the protein. Newborn rabbit heart cDNAs, obtained using the reverse-transcriptase polymerase chain reaction (RT-PCR), were amplified using primers derived from this cDNA. Three full-length cDNAs that differed by the inclusion or exclusion of three short nucleotide sequences within the cDNAs were obtained. Amplification in the 5′ half of the cDNAs confirmed that multiple cTnT products arose because of the variable inclusion of an 18- and a 30-nt sequence. The 30-nt sequence has homology with previously described alternatively spliced exons in rat and chicken cTnT, whereas the 18-nt sequence has not been described previously. RT-PCR in the 3′ half of the cDNAs confirmed an additional region of heterogeneity: the presence, in part or in full, or absence of a 9-nt region, which matches the alternatively spliced exon 12 described for rat cTnT. In vitro transcription and translation of four cDNA clones containing both the 18- and 30-nt sequences, the 30-nt sequence, the 18-nt sequence, or neither generated protein isoforms that comigrated with cTnT1, cTnT2, cTnT3, and cTnT4, respectively. Polyclonal antisera, raised against the peptide encoded by the 30-nt region, reacted only with cTnT1and cTnT2. These results demonstrate that alternative independent splicing of two exons in the NH2-terminal half of the cTnT coding region yields four rabbit cTnT isoforms, and an area of heterogeneity in the C-terminal half provides the potential for 12 isoforms. The previously demonstrated positive correlation between the relative amount of cTnT2and the sensitivity of those myofilaments to calcium suggests that the 10-residue peptide encoded by the 30-nt exon alters thin-filament regulation of contraction.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

The presence of chloride-formate anion exchange in vascular smooth muscle cells (VSMCs) and cardiac myocytes was investigated. Imposing an outward chloride gradient in sarcolemmal microsomes isolated from canine aorta stimulated [14C] formate uptake compared with the absence of a chloride gradient (24.3±2.33 versus 9.8±1.41 pmol/mg protein for 30 seconds,P<.03) and induced transient uphill [14C] formate uptake. The chloride-formate exchange was significantly inhibited in the presence of 1 mmol/L 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) or furosemide (57% and 61%, respectively). Incubation of rat cultured VSMCs in a medium containing [14C] formate resulted in uptake of formate that was significantly DIDS and furosemide sensitive (79.34 ±2.47, 43.03±2.37, and 44.65±1.68 pmol/mg protein for 4 minutes in control, DIDS, and furosemide groups, respectively). Preincubation of the VSMCs in chloride-free medium significantly reduced the DIDS-sensitive (36.31 versus 16.85 pmol/mg protein for 4 minutes,P< .001) and furosemide-sensitive (34.72 versus 8.78 pmol/mg protein for 4 minutes,P<.001) [14C] formate uptake. These results are compatible with the presence of chloride-formate exchange in VSMCs. Influx of [14C] formate into sarcolemmal vesicles isolated from canine heart was significantly higher in the presence of an outward chloride gradient than in its absence (18.1 ±2.3 versus 9.6±1.7 pmol/mg protein for 30 seconds,P<.03). The chloride-formate exchange was significantly inhibited in the presence of 1 mmol/L DIDS or furosemide (41% and 52%, respectively). We conclude that the distribution of chloride-formate exchange may be more universal than previously suggested. The physiological significance of this anion exchanger remains to be determined.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

Tissue factor (TF) is a transmembrane protein that binds factor VII/VIIa, thus activating the extrinsic blood coagulation pathway. Since this pathway appears to be involved in the formation of intravascular thrombi, the anti-rabbit TF monoclonal antibody, AP-1, was produced and tested as an antithrombotic agent in a rabbit model of recurrent intravascular thrombosis. In this model, a plastic constrictor is positioned around the injured rabbit carotid arteries, and flow is monitored with a Doppler flow probe. This produces cyclic flow variation (CFV) in the carotid artery, which is caused by recurrent formation and dislodgment of thrombi at the site of the stenosis. After monitoring CFV pattern for 30 minutes, AP-1 was infused intravenously into nine rabbits at doses of 0.05 to 1.5 mg/kg body weight, and a control monoclonal antibody that does not react with rabbit TF was infused into four additional rabbits. In all rabbits receiving AP-1, CFV was abolished, and a steady normal blood flow was restored, indicating that thrombus formation had been blocked by AP-1. By contrast, in all rabbits that received the control monoclonal antibody, CFV continued unaltered. There was no change in the partial thromboplastin time and ex vivo platelet aggregation to several different agonists after infusion of AP-1, indicating an absence of systemic effects on the coagulation process. We conclude that activation of the extrinsic coagulation pathway has a key role in triggering intravascular thrombosis and that an anti-TF monoclonal antibody is an effective antithrombotic agent that could have therapeutic potential for humans.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

It is not known whether myocardial energy requirements can be increased to the degree that they exceed myocardial O2availability in the absence of abnormalities of coronary blood flow or coronary reserve. To determine whether this form of “demand ischemia” occurs, 10 swine were subjected to pressure overload induced by aortic constriction, inotropic and chronotropic stimulation by dobutamine, and the combination of these interventions. In an additional 9 animals, intravenous adenosine was administered during the combination of constriction and dobutamine to determine whether further increases in coronary flow could be achieved and if they would attenuate the metabolic changes. Left ventricular anterior wall transmural blood flow was measured by radioactive microspheres. Energy phosphates were assessed by31P magnetic resonance spectroscopy using the Fourier series window technique to increase the proportion of signal derived from the subendocardium. Myocardial lactate release was quantified independent of net lactate uptake using an isotopic tracer technique. The three interventions produced 39% to 195% increases in myocardial O2uptake from control measurements. The phosphocreatine to ATP ratio (PCr/ATP), uncorrected for partial saturation, fell significantly, from 1.39±0.10 at control conditions to 1.25±0.10 with dobutamine alone and 1.15±0.08 with dobutamine plus constriction (P<.05 for both). Myocardial lactate release rose from 0.21±0.03 mol · g−1· min−1at control conditions to 0.45±0.05 and 0.59±0.10 μmol g−1· min−1, respectively (P<.05 for both), with these two interventions. Although transmurally averaged left ventricular blood flow rose from 0.97±0.09 mL·g−1· min−1at control conditions to 3.25±0.47 mL · g−1· min−1(P<.001) and subendocardial blood flow increased from 1.02±0.09 to 2.92±0.45 mL· g−1· min−1(P<.001) at the highest of the three increased work states, the subendocardial to subepicardial flow ratio declined progressively from 1.13±0.08 to 0.87±0.04 (P<.05). With a further increase in aortic constriction, myocardial 02 uptake and subepicardial blood flow rose, whereas subendocardial blood flow did not change, and there was a further decline in PCr/ATP and a rise in lactate release. Although adenosine increased the average myocardial blood flow during high work state from 3.79±0.91 to 6.29±1.08 mL· g−1· min−1(P<.001), the further rise in subendocardial flow from 3.08±0.62 to 3.78±0.68 mL· g−1· min−1was not significant, nor were the accompanying changes in PCr/ATP or lactate metabolism. The progressive decline in the subendocardial to subepicardial ratio and PCr/ATP with a concomitant increase in myocardial lactate release is consistent with the occurrence of demand ischemia in the porcine left ventricle during high work states, and the lack of a significant increase in subendocardial blood flow or attenuation of the metabolic changes suggests limitation of subendocardial coronary reserve under these conditions.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

A role for the internal elastic lamina (IEL), which separates the intima and media of an artery wall, as a restrictive barrier to macromolecular movement has been suggested in atherosclerotic lesion development or restenosis during angioplasty. The permeability coefficient of the IEL, however, has never been quantified in unperturbed vessels in vivo. Using a newly developed technique, we measured the concentration distributions in both intima and media of cationic (pI ≈ 8.5) and anionic (pI ≈ 6.3) isozymes of the 44-kD macromolecule horseradish peroxidase (HRP). Two mathematical models of arterial wall transport differing in their resolution of the intima were required to simulate the concentration distribution data and to estimate the parameters of interest. Optimal estimates of the permeability coefficients of the endothelium (PE) and IEL (PIEL) to HRP were determined by the best least-squares fit of the two models to experimental data. These estimates (anionic: PE=0.050±0.021 μm/min, PIEL= 0.146±0.082 μm/min, n = 8; cationic: PE= 0.034 ± 0.018 μm/min, PIEL= 0.110±0.047 μm/min, n = 8) indicate that the IEL is responsible for ≈25% (anionic, 26±9%; cationic, 25±13%) of the resistance to HRP transport from the blood into the arterial media. Although both parameters were less for the cationic preparation, the differences were not significant, and the relative role of the IEL was similar for both molecules. These data demonstrate the importance of the IEL in controlling the intimal accumulations of plasma-borne macromolecules, and they imply a role for the IEL in influencing paracrine communication between cells of the intima and media.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID

摘要:

Long-chain acylcarnitines (LCACs) increase rapidly within minutes after the onset of ischemia in vivo or hypoxia in vitro and produce a time-dependent reversible reduction in gap junctional conductance in isolated myocyte pairs. The present study was performed to assess whether LCACs contribute to cellular uncoupling in response to ischemia in isolated blood perfused rabbit papillary muscles by use of simultaneous measurements of transmembrane action potentials, extracellular electrograms, extracellular K+, and tissue LCACs and ATP. LCACs increased threefold in response to 20 minutes of no-flow ischemia from 127±5 to 397±113 pmol/mg protein (P<.01), concomitant with the onset of cellular uncoupling, extracellular K+accumulation, and a marked reduction in conduction velocity and action potential duration. To assess whether inhibition of the accumulation of LCACs modified the electrophysiological alterations during ischemia, muscles were pretreated with either sodium 2-(5-(4-chlorophenyl)-pentyl)-oxirane-2-carboxylate (POCA, 10 μmol/L) or oxfenicine (100 μmol/L), inhibitors of carnitine acyltransferase I. Both POCA and oxfenicine completely prevented the increase in LCACs even with 40 minutes of ischemia (138±37 and 56±4 pmol/mg protein, respectively), associated with a marked delay in the onset and progression of cellular uncoupling and ischemic contracture. Although POCA and oxfenicine did not affect either the initial early rise in extracellular K+or the initial fall in conduction velocity, both agents markedly delayed the secondary rise in extracellular K+as well as the secondary fall in conduction velocity, independent of the level of tissue ATP. Thus, LCACs accumulate during myocardial ischemia and contribute substantially to the initiation of cell-to-cell uncoupling. Inhibition of carnitine acyltransferase I and prevention of the increase in LCACs markedly delays cellular uncoupling and development of ischemic contracture in response to ischemia.

ISSN:0009-7330    

出版商:OVID    

年代:1994

数据来源: OVID