摘要:

Membrane currents from single smooth muscle cells enzymatically isolated from canine renal artery were recorded using the patch-clamp technique in the whole-cell and cell-attached configurations. These cells exhibited a mean resting potential, input resistance, membrane time constant, and cell capacitance of −51.8±2.1 mV, 5.2±0.98GΩ, 116.2±16.4 msec, and 29.1±2.0 pF, respectively. Inward current, when elicited from a holding potential of —80 mV, activated near —50 mV, reached a maximum near 0 mV and was sensitive to the dihydropyridine agonist Bay K 8644 and dihydropyridine antagonist nisoldipine. Two components of macroscopic outward current were identified from voltage-step and ramp depolarizations. The predominant charge carrier of the net outward current was identified as K+by tail-current experiments (reversal potential, −61.0±0.8 mV in 10.8 mM [K+]o/140 mM [K+]i). The first component was a small, low-noise, voltage- and time-dependent current that activated between —40 and —30 mV (IK(dr)), and the second component was a larger, noisier, voltage- and time-dependent current that activated at potentials positive to +10 mV (IK(Ca)). Both IK(dr)and lK(Ca)displayed little inactivation during long (4-second) voltage steps. IK(Ca)and lK(dr)could be pharmacologically separated by using various Ca2' and K+channel blockers. IK(Ca)was substantially inhibited by external NiCI2(500 μM), CdCI2(300 μM), EGTA (5 mM), tetraethylammonium (Kiat +60 mV, 307 μM), and charybdotoxin (100 nM) but was insensitive to 4-aminopyridine (0.1–10 mM). IK(dr)was inhibited by 4-aminopyridine (Kiat +10 mV, 723 μM) and tetraethylammonium (Kiat +10 mV, 908 μM) but was insensitive to external NiCI2(500 μM), CdCI2(300 μM), EGTA (5 mM), and charybdotoxin (100 nM). Two types of single K+channels were identified in cell-attached patches. The most abundant K+channel that was recorded exhibited voltage-dependent activation, was blocked by external tetraethylammonium (250 μM), and had a large single-channel conductance (232±f12 pS with 150 mM K+in the patch pipette, 130±17 pS with 5.4 mM K+in the patch pipette). The second channel was also voltage dependent, was blocked by 4-aminopyridine (5 mM), and exhibited a smaller single-channel conductance (104±8 pS with 150 mM K+in the patch pipette, 57±6 pS with 5.4 mM K+in the patch pipette). These results suggest that depolarization of canine renal artery cells opens dihydropyridine-sensitive Ca2+channels and at least two K+channels. The two time-dependent K+currents (IK(Ca)and IK(dr)) reflect the behavior of two distinct K' channels. According to the pharmacology of the whole-cell and single-channel experiments, IK(Ca)is primarily carried by the large conductance Ca2+-activated K+channel, and IK(dr)is carried by the smaller conductance delayed rectifier K' channel.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

Smooth muscle cells (SMCs) cultured from the neointima of injured rat carotid arteries have a different shape and organization in vitro than SMCs from the uninjured media. The morphology of neointimal SMCs from adult rats strongly resembles that of a subset of medial SMCs from 12-day-old rat pups. In the present study, we show that adult carotid neointimal SMCs in vitro express the platelet-derived growth factor (PDGF)-B gene but have little or no PDGF a-receptor mRNA. In contrast, medial SMCs from contralateral uninjured carotids, grown and passaged under identical conditions, contain abundant PDGF α-receptor mRNA but little or no PDGF-B mRNA. Transcript levels for PDGF-A or PDGF α-receptor were not different in neointimal versus medial SMC cultures. The PDGF mRNA phenotype of adult neointimal SMCs strongly resembles that of an aortic medial SMC subset from newborn rat pups. Although intriguing, the differences in SMC phenotypes we observed in cell culture may depend on unique conditions in vitro and do not necessarily mean that analogous SMC diversity also exists in vivo. To address this question, we constructed and screened a SMC cDNA library for additional molecular markers of the common “pup-intimal” SMC phenotype. Two cDNA clones were identified whose cognate mRNA levels were developmentally regulated in rat aorta in vivo and were present at high levels in the adult carotid neointima formed 2 weeks after balloon catheter injury. Importantly, elevated levels of these two cognate mRNAs in carotid neointima compared with underlying media were maintained in cultures of neointimal versus medial SMCs in vitro. DNA sequence analysis indicated that the cDNA clones encoded rat tropoelastin and SS1procollagen (type I). These results provide further evidence that neointima formation in the adult rat carotid artery depends on reexpression of an SMC phenotype or subpopulation with special properties characteristic of earlier stages of artery wall development. Our studies to date indicate that two of these special properties are paracrine growth factor production and extracellular matrix synthesis.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

In 36 normolipemic pigs randomized to a 4-week feeding with regular pig chow (n=18, control group) or chow supplemented with cod liver oil (1 ml/kg per day) (n=18, treated group), treatment with cod liver oil produced a significant decrease in serum cholesterol, low density lipoprotein cholesterol, and triglycerides. Deep carotid arterial wall injury (media exposed) by balloon angioplasty was associated with less111In-labeled platelet deposition (24.6±4.8×106/cm2versus 62.5±17.0×106/cm2, p<0.05; difference, −33.8×106/cm2; 95% confidence interval [CI], −1.9×106/cm2to −73.9×106/cm2) and injury-related vasoconstriction (21.3±2.2% versus 30.9±2.9o,p<0.05; difference, −9.6%; 95% CI, −2.2% to −17.00) in the cod liver oil-treated group than in the control group; with mild injury (media not exposed), platelet deposition was low and unchanged (6.2±0.5×106/cm2versus 7.8±0.7×106/cm2; difference, −1.6×106/ cm2; 95% CI, −1.1×106/cm2to +4.3×106/cm2), but associated vasoconstriction was reduced respectively (16.3±2.0% versus 23.0±2.2%,p<0.05; difference, −6.7%; 95% CI, −0.6% to −12.8%). When arterial blood from cod liver oil-treated pigs superfused normal aortic media ex vivo, platelet deposition onto the normal aortic media was lower than when arterial blood from control pigs superfused the normal aortic media (43.7±8.8×106/cm2versus 66.8±13.0×106/cm2,p<0.05). A mild increase in the eicosapentaenoic acid/arachidonic acid ratio to 0.5 or greater was associated with a significant reduction in platelet deposition and vasoconstriction, whereas a ratio in excess of 2.0 was needed to significantly lower serum lipid levels. In addition, there were no correlations between serum cholesterol and platelet deposition or vasoconstriction, making it unlikely that the modest reduction in serum lipids would influence the acute platelet-arterial wall interactions. These beneficial effects of cod liver oil on platelet-arterial wall interactions, occurring with only a mild increase in the eicosapentaenoic acid/arachidonic acid ratio, may have implications for thrombosis and vasospasm after deep arterial injury.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

Hypertension is a risk factor for coronary atherosclerosis possibly via an adverse effect on the vascular endothelium. Endothelium-mediated relaxation is impaired in animal models of hypertension. However, the effects of hypertension on human coronary artery endothelial cell function are unknown. To test whether endothelium-mediated relaxation is impaired in the coronary arteries of patients with hypertension, we studied 14 patients with essential hypertension requiring therapy and 15 nonhypertensive control patients undergoing cardiac catheterization. All had angiographically normal, smooth-appearing coronary arteries. Patients were matched for age and other coronary atherosclerosis risk factors. To assess endothelial cell function, the endothelium-dependent vasodilator acetylcholine (ACh, 0.01, 0.1, and 1.0 μM) and the endothelium-independent vasodilator nitroglycerin (40 μg) were selectively infused into the left anterior descending or circumflex coronary artery. Diameter change (expressed as percent) was assessed using quantitative angiography. There was a marked vasoconstrictor response to serial doses of ACh in hypertensive patients (−7%, −21%, and −27%) compared with control patients (−4%, −5%, and −7%) (p<0.02). The vasodilator response to nitroglycerin was preserved in hypertensive patients (+29%1) and control patients (+25%) (p=NS), suggesting that endothelial cell dysfunction accounted for the differences in response to ACh. Thus, patients with hypertension have an accentuated coronary vasoconstrictor response to ACh, suggesting that endothelium-mediated regulation of coronary vascular tone is impaired by essential hypertension. This may reflect more generalized coronary endothelial changes contributing to the pathogenesis of atherosclerosis as well as hypertension.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

Newly synthesized heparan sulfates purified from the cell layer of bovine aortic endothelial cells (BAECs) and main pulmonary artery endothelial cells (BPAECs) cultured under either normoxic (21% oxygen) or hypoxic (3% oxygen) conditions were characterized by size, charge, and capacity to bind to antithrombin III. Incorporation of radiolabeled sulfate into cell layer-associated heparan sulfate was reduced by 70% in BAECs and by 45% in BPAECs during exposure to 3% oxygen; degradation of radiolabeled heparan sulfate was not affected by hypoxia. However, the percentage of total radiolabeled heparan sulfate that bound to antithrombin III was increased by 33% for BAECs and by 120% for BPAECs when compared with radiolabeled heparan sulfate synthesized during the 21% oxygen exposure. Both the high- and low-antithrombin III affinity radiolabeled heparan sulfate consisted of two components of different sizes; the low-affinity components (mean sizes, 60 and 40 kd) generated under normoxic conditions were smaller than their respective high-affinity components (mean sizes, 70 and 55 kd) by molecular sieve chromatog-raphy. The components of low-antithrombin III affinity heparan sulfate generated during exposure to 3% oxygen were increased in size compared with the corresponding low-affinity components generated during the 21% oxygen exposure for both BPAECs and BAECs. In addition, the amount of the larger high-antithrombin III affinity component was reduced in both cell types exposed to hypoxia. There was no difference in functional heparin-like activity per dish between cells cultured at 3% and 21% oxygen; BAECs had twofold to threefold greater activity per dish than did BPAECs at both levels of oxygen. We conclude that the overall amounts of heparan sulfates synthesized by cultured BPAECs and BAECs are affected similarly but not to the same extent during exposure to 3% oxygen and that hypoxia may differentially influence the chain length and antithrombin III-binding capacity of heparan sulfate species.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

The aims of the present study were to determine the response of rat cremaster muscle first-order arterioles to hypoxia and the role of endothelium-derived prostaglandins in the response. Isolated arterioles were cannulated, pressurized to 65 mm Hg, and studied in a no-flow condition in a bath containing Krebs' bicarbonate solution, pH 7.4, equilibrated with 21% O2-5% CO2-74% N2(Po2,150 mm Hg) or 95% N2-5% CO2(Po2,15 mm Hg [hypoxia]). Responses to hypoxia and vasoactive substances were studied before and after removal of the endothelium or blockade of prostaglandin synthesis by the administration of indomethacin (10-5M). Addition to the suffusion solution of arachidonic acid (10-7and 10-6M), prostaglandin E2(10-9and 10-8M), acetylcholine (10-8and 10-6M), or sodium nitroprusside (10-8M) evoked significant arteriolar dilation. When the bath Po2was reduced from 150 to 15 mm Hg, arteriolar diameters increased by 58.8±9.3 μm (61%). Removal of the endothelium completely inhibited responses to hypoxia, acetylcholine, and arachidonic acid, whereas responses to sodium nitroprusside and prostaglandin E2remained unaltered. In arterioles with an intact endothelium, indomethacin completely inhibited the responses to hypoxia and arachidonic acid, whereas responses to acetylcholine and sodium nitroprusside were unaltered. These findings support the conclusion that endothelium-derived prostaglandins mediate the arteriolar dilation to hypoxia in rat skeletal muscle arterioles.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

To examine the cellular mechanisms of contractile dysfunction in postinfarction heart failure, we studied the effects of β-adrenergic receptor stimulation on contractile function and Ca2+ihandling of nonin-farcted papillary muscles from sham-operated (n=17) and infarcted (n=17) rats. Ca2+itransients 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<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+iIn muscles from sham-operated rats, isoproterenol increased both the amplitude of the Ca2+itransient 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+itransient. Isoproterenol abbreviated the time course of the isometric twitch and the Ca2+itransient 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+ihandling. In addition, compared with muscles from untreated infarcted rats, muscles from the captopril-treated rats exhibited improved contractile responses to increasing [Ca2+]oor isoproterenol. The inotropic response to isoproterenol in muscles from all three groups of rats had a significant negative correlation (r=−0.64, p<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.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

Muscarinic K+channels in inside-out patches of atrial cells from guinea pig or rabbit can be activated by Mg2+-ATP in the absence of acetylcholine and GTP or GDP. The ATP-dependent activation involves a phosphorylation and is postulated to be due to the association of a membrane-bound nucleoside diphosphate kinase (NDPK) with the G protein GK: direct phosphorylation of the GK-bound GDP into GTP, catalyzed by NDPK, would result in activation of the G protein and, hence, activation of the channels. The aim of this study was to identify the presence of NDPK activity in atrial membranes by investigating the phosphate transfer between tritium-labeled nucleotides. We show that frog, guinea pig, and human atrial membranes contain a substantial NDPK activity since they catalyze the conversion from [3H]GDP+nucleoside triphosphate (NTP or NTPγS) to [3H]GTP (or [3H]GTPγS), from [3H]ADP+NTP to [3H]ATP, and from [3H]GTP+nucleoside diphosphate (NDP) to [3H]GDP. The phosphate transfer rates for the [3H]GDP+ATP to [3H]GTP conversion are 1.8, 0.5, and 2.4 μmol inorganic phosphate formation/mg per 10 minutes at 37°C in frog, guinea pig, and human, respectively. The order of substrate efficiency for different NTPs was ATP>ITP≅GTP>UTP>CTP, which parallels the effliciency of these nucleotides in their activation of the muscarinic K+channels. Addition of other nucleotides blocked the transphosphorylation reaction, indicating that the NTP-NDP conversion mechanism is aspecific, as is expected for an NDPK-catalyzed reaction. In conclusion, the data support the concept of NDPK involvement in the atrial muscarinic signal transduction cascade.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

Inhibition of aerobic metabolism leads to a major disruption of cardiac cell homeostasis. The purpose of the present study was twofold: 1) We determined the relative importance of junctional and nonjunctional membrane resistance (Rjand Rm, respectively) in the development of propagation failure during inhibition of aerobic metabolism in guinea pig ventricular cell pairs. 2) We used the patch-action potential clamp technique in single ventricular myocytes to study some of the properties of the membrane channels that are responsible for shortening of action potential duration and eventual failure of cell excitation after metabolic blockade. In most experiments, whole-cell patch pipettes were filled with a solution containing 1 mM EGTA, 5 mM HEPES, and 5 mM ATP. Our results in cell pairs showed that pharmacological inhibition of aerobic metabolism with the mitochondrial uncoupler 2,4-dinitrophenol (DNP) led to a drop in Rmfollowed by an increase in Rj. The increase in Rjwas not sufficient to cause a measurable delay in cell-to-cell propagation, whereas the drop in R. consistently led to failure of cell excitation. Similar results were obtained in additional experiments in which the EGTA concentration in the pipette was reduced to 50 μM. Similar results were also obtained by loading the recording patch pipettes with a solution containing only 0.1 mM ATP. Our patch-action potential clamp experiments, on the other hand, revealed that DNP induced the opening of time- and voltage-independent membrane channels, with a unitary conductance of 23 pS. The channels allowed for the passage of outward current in the voltage range of the action potential, and the increase in membrane patch conductance correlated with the observed shortening of action potential duration during DNP superfusion. Our experiments provide the first simultaneous recordings of action potentials and DNP-induced channel currents in guinea pig ventricular myocytes. Overall, the data provide new evidence for the understanding of the cellular and subcellular mechanisms involved in the development of slow conduction velocity and propagation block after metabolic blockade.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID

摘要:

The regulatory effects of thyroid hormone on biosynthesis of myocardial proteins that originate from cardiac myocytes are well established. Little is known, however, of regulatory effects of thyroid hormone on interstitial proteins. In this study we examined the effects of thyroid hormone on collagen gene expression in thyroid hormone-induced myocardial hypertrophy and the response of cardiac fibroblasts to thyroid hormone in culture. Adult male Sprague-Dawley rats were treated intraperitoneally with L-thyroxin (10 μg/100 g body wt) for 2 hours or 1, 2, 3, 6, 12, or 14 days. Northern blot analysis of RNA from total ventricular tissue showed that after 2 hours of treatment, the abundance of mRNA for pro α2(I) collagen decreased by 53% (p<0.05) and reached the lowest level (60% decrease, p<0.02) at day 1, remained diminished at day 3, and then gradually returned toward normal levels. After transient transfection of chimeric DNA containing collagen type I promoter-chloramphenicol acetyl transferase (CAT) gene into the thyroxin-treated cardiac fibroblasts, the level of CAT activity decreased significantly. Treatment of cardiac fibroblasts in culture (10 nM l-thyroxin) resulted in a 33% (p< 0.005) decrease in the abundance of mRNA for pro α2(I) collagen. The stability of the mRNA for pro α2(I) collagen in cardiac fibroblasts, as measured by mRNA half-life, was slightly (16.6%) decreased by thyroid-hormone treatment. Collagen synthesis as shown by immunofluorescent staining of intracellular collagen in cultured fibroblasts and in frozen sections of myocardium was also diminished. Interestingly, the abundance of mRNA for transforming growth factor-β1in the myocardium increased by 53% (p<0.05) within hours and reached a peak (75% increase,p< 0.001) at day 1 after treatment. The abundance of mRNA for transforming growth factor-β1was also increased (94%,p< 0.005) in cardiac fibroblasts after treatment with l-thyroxin. Thyroxin treatment of cardiac fibroblasts led to the induction of proto-oncogenes c-fosand c-junand early growth response gene within 1 hour. Treatment of cardiac fibroblasts with l-thyroxin led to a 357% (p< 0.001) increase in [3H]thymidine incorporation into the cell nuclei compared with that in untreated cells. Together, these findings suggest that cardiac fibroblasts and proteins originating from them are targets for thyroid hormone action in the myocardium and that this hormone plays a regulatory role on interstitial protein gene expression. Data further suggest that thyroid hormone-induced myocardial hypertrophy could be distinguished by the lack of cardiac fibrosis.

ISSN:0009-7330    

出版商:OVID    

年代:1992

数据来源: OVID