Vitronectin inhibits the thrombotic response to arterial injury in mice.
(17/7624)
Vitronectin (VN) binds to plasminogen activator inhibitor-1 (PAI-1) and integrins and may play an important role in the vascular response to injury by regulating fibrinolysis and cell migration. However, the role of VN in the earliest response to vascular injury, thrombosis, is not well characterized. The purpose of this study was to test the hypothesis that variation in vitronectin expression alters the thrombotic response to arterial injury in mice. Ferric chloride (FeCl3) injury was used to induce platelet-rich thrombi in mouse carotid arteries. Wild-type (VN +/+, n = 14) and VN-deficient (VN -/-, n = 15) mice, matched for age and gender, were studied. Time to occlusion after FeCl3 injury was determined by application of a Doppler flowprobe to the carotid artery. Occlusion times of VN -/- mice were significantly shorter than those of VN +/+ mice (6.0 +/- 1.2 minutes v 17.8 +/- 2.3 minutes, respectively, P < .001). Histologic analysis of injured arterial segments showed that thrombi from VN +/+ and VN -/- mice consisted of dense platelet aggregates. In vitro studies of murine VN +/+ and VN -/- platelets showed no significant differences in ADP-induced aggregation, but a trend towards increased thrombin-induced aggregation in VN -/- platelets. Purified, denatured VN inhibited thrombin-induced platelet aggregation, whereas native VN did not. Thrombin times of plasma from VN -/- mice (20.5 +/- 2.1 seconds, n = 4) were significantly shorter than those of VN +/+ mice (34.2 +/- 6.7 seconds, n = 4, P < .01), and the addition of purified VN to VN -/- plasma prolonged the thrombin time into the normal range, suggesting that VN inhibits thrombin-fibrinogen interactions. PAI-1-deficient mice (n = 6) did not demonstrate significantly enhanced arterial thrombosis compared with wild-type mice (n = 6), excluding a potential indirect antithrombin function of VN mediated by interactions with PAI-1 as an explanation for the accelerated thrombosis observed in VN -/- mice. These results suggest that vitronectin plays a previously unappreciated antithrombotic role at sites of arterial injury and that this activity may be mediated, at least in part, by inhibiting platelet-platelet interactions and/or thrombin procoagulant activity. (+info)
Shrinkage-induced activation of Na+/H+ exchange in rat renal mesangial cells.
(18/7624)
Using the pH-sensitive dye 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), we examined the effect of hyperosmolar solutions, which presumably caused cell shrinkage, on intracellular pH (pHi) regulation in mesangial cells (single cells or populations) cultured from the rat kidney. The calibration of BCECF is identical in shrunken and unshrunken mesangial cells if the extracellular K+ concentration ([K+]) is adjusted to match the predicted intracellular [K+]. For pHi values between approximately 6.7 and approximately 7.4, the intrinsic buffering power in shrunken cells (600 mosmol/kgH2O) is threefold larger than in unshrunken cells (approximately 300 mosmol/kgH2O). In the nominal absence of CO2/HCO-3, exposing cell populations to a HEPES-buffered solution supplemented with approximately 300 mM mannitol (600 mosmol/kgH2O) causes steady-state pHi to increase by approximately 0.4. The pHi increase is due to activation of Na+/H+ exchange because, in single cells, it is blocked in the absence of external Na+ or in the presence of 50 microM ethylisopropylamiloride (EIPA). Preincubating cells in a Cl--free solution for at least 14 min inhibits the shrinkage-induced pHi increase by 80%. We calculated the pHi dependence of the Na+/H+ exchange rate in cell populations under normosmolar and hyperosmolar conditions by summing 1) the pHi dependence of the total acid-extrusion rate and 2) the pHi dependence of the EIPA-insensitive acid-loading rate. Shrinkage alkali shifts the pHi dependence of Na+/H+ exchange by approximately 0.7 pH units. (+info)
Synthesis and characterization of dual-wavelength Cl--sensitive fluorescent indicators for ratio imaging.
(19/7624)
The fluorescence of quinolinium-based Cl- indicators such as 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) is quenched by Cl- by a collisional mechanism without change in spectral shape. A series of "chimeric" dual-wavelength Cl- indicators were synthesized by conjugating Cl--sensitive and -insensitive chromophores with spacers. The SPQ chromophore (N-substituted 6-methoxyquinolinium; MQ) was selected as the Cl--sensitive moiety [excitation wavelength (lambdaex) 350 nm, emission wavelength (lambdaem) 450 nm]. N-substituted 6-aminoquinolinium (AQ) was chosen as the Cl--insensitive moiety because of its different spectral characteristics (lambdaex 380 nm, lambdaem 546 nm), insensitivity to Cl-, positive charge (to minimize quenching by chromophore stacking/electron transfer), and reducibility (for noninvasive cell loading). The dual-wavelength indicators were stable and nontoxic in cells and were distributed uniformly in cytoplasm, with occasional staining of the nucleus. The brightest and most Cl--sensitive indicators were alpha-MQ-alpha'-dimethyl-AQ-xylene dichloride and trans-1, 2-bis(4-[1-alpha'-MQ-1'-alpha'-dimethyl-AQ-xylyl]-pyridinium)ethyl ene (bis-DMXPQ). At 365-nm excitation, emission maxima were at 450 nm (Cl- sensitive; Stern-Volmer constants 82 and 98 M-1) and 565 nm (Cl- insensitive). Cystic fibrosis transmembrane conductance regulator-expressing Swiss 3T3 fibroblasts were labeled with bis-DMXPQ by hypotonic shock or were labeled with its uncharged reduced form (octahydro-bis-DMXPQ) by brief incubation (20 microM, 10 min). Changes in Cl- concentration in response to Cl-/nitrate exchange were recorded by emission ratio imaging (450/565 nm) at 365-nm excitation wavelength. These results establish a first-generation set of chimeric bisquinolinium Cl- indicators for ratiometric measurement of Cl- concentration. (+info)
Enteropathogenic E. coli attenuates secretagogue-induced net intestinal ion transport but not Cl- secretion.
(20/7624)
Enteric bacterial pathogens often increase intestinal Cl- secretion. Enteropathogenic Escherichia coli (EPEC) does not stimulate active ion secretion. In fact, EPEC infection decreases net ion transport in response to classic secretagogues. This has been presumed to reflect diminished Cl- secretion. The aim of this study was to investigate the influence of EPEC infection on specific intestinal epithelial ion transport processes. T84 cell monolayers infected with EPEC were used for these studies. EPEC infection significantly decreased short-circuit current (Isc) in response to carbachol and forskolin, yet 125I efflux studies revealed no difference in Cl- channel activity. There was also no alteration in basolateral K+ channel or Na+-K+-2Cl- cotransport activity. Furthermore, net 36Cl- flux was not decreased by EPEC. No alterations in either K+ or Na+ transport could be demonstrated. Instead, removal of basolateral bicarbonate from uninfected monolayers yielded an Isc response approximating that observed with EPEC infection, whereas bicarbonate removal from EPEC-infected monolayers further diminished Isc. These studies suggest that the reduction in stimulated Isc is not secondary to diminished Cl- secretion. Alternatively, bicarbonate-dependent transport processes appear to be perturbed. (+info)
Nitric oxide-mediated regulation of transepithelial sodium and chloride transport in murine nasal epithelium.
(21/7624)
Transepithelial ion transport is regulated by a variety of cellular factors. In light of recent evidence that nitric oxide (NO) production is decreased in cystic fibrosis airways, we examined the role of NO in regulating sodium and chloride transport in murine nasal epithelium. Acute intervention with the inducible NO synthase (iNOS)-selective inhibitor S-methylisothiourea resulted in an increase of amiloride-sensitive sodium absorption observed as a hyperpolarization of nasal transepithelial potential difference. Inhibition of iNOS expression with dexamethasone also hyperpolarized transepithelial potential difference, but only a portion of this increase proved to be amiloride sensitive. Chloride secretion was significantly inhibited in C57BL/6J mice by the addition of both S-methylisothiourea and dexamethasone. Mice lacking iNOS expression [NOS2(-/-)] also had a decreased chloride-secretory response compared with control mice. These data suggest that constitutive NO production likely plays some role in the downregulation of sodium absorption and leads to an increase in transepithelial chloride secretion. (+info)
Delayed rectifier potassium current in undiseased human ventricular myocytes.
(22/7624)
OBJECTIVE: The purpose of the study was to investigate the properties of the delayed rectifier potassium current (IK) in myocytes isolated from undiseased human left ventricles. METHODS: The whole-cell configuration of the patch-clamp technique was applied in 28 left ventricular myocytes from 13 hearts at 35 degrees C. RESULTS: An E-4031 sensitive tail current identified the rapid component of IK (IKr) in the myocytes, but there was no evidence for an E-4031 insensitive slow component of IK (IKs). When nifedipine (5 microM) was used to block the inward calcium current (ICa), IKr activation was fast (tau = 31.0 +/- 7.4 ms, at +30 mV, n = 5) and deactivation kinetics were biexponential and relatively slow (tau 1 = 600.0 +/- 53.9 ms and tau 2 = 6792.2 +/- 875.7 ms, at -40 mV, n = 7). Application of CdCl2 (250 microM) to block ICa altered the voltage dependence of the IKr considerably, slowing its activation (tau = 657.1 +/- 109.1 ms, at +30 mV, n = 5) and accelerating its deactivation (tau = 104.0 +/- 18.5 ms, at -40 mV, n = 8). CONCLUSIONS: In undiseased human ventricle at 35 degrees C IKr exists having fast activation and slow deactivation kinetics; however, there was no evidence found for an expressed IKs. IKr probably plays an important role in the frequency dependent modulation of repolarization in undiseased human ventricle, and is a target for many Class III antiarrhythmic drugs. (+info)
Alterations of cross-bridge kinetics in human atrial and ventricular myocardium.
(23/7624)
CONDENSED ABSTRACT: We analyzed actomyosin cross-bridge kinetics in human atrial and ventricular muscle strip preparations by using sinusoidal length changes from 0.1 to 60 Hz. The minimum stiffness frequency was higher in atrial than in ventricular human myocardium and lower in failing than in non-failing left ventricular human myocardium. beta-Adrenergic stimulation increased the minimum stiffness frequency by 18 +/- 3% (p < 0.05). Cross-bridge kinetics are temperature-dependent, with a Q10 of at least 2.7. BACKGROUND: Dynamic stiffness measurements have revealed acute and chronic alterations of actomyosin cross-bridge kinetics in cardiac muscles of a variety of different animal species. We studied dynamic stiffness in right atrial and left ventricular preparations of non-failing and failing human hearts and tested the influence of the temperature and beta-adrenergic stimulation on cross-bridge kinetics. METHODS AND RESULTS: Muscle strips were prepared from right atria and left ventricles from human non-failing and failing hearts. After withdrawal of calcium, steady contracture tension was induced by the addition of 1.5 mM barium chloride. Sinusoidal length oscillations of 1% muscle length were applied, with a frequency spectrum of between 0.1 and 60 Hz. Dynamic stiffness was calculated from the length change and the corresponding force response amplitude. The specific minimum stiffness frequency, which indicates the interaction between cross-bridge recruitment and cross-bridge cycling dynamics, was analyzed for each condition: (1) The minimum stiffness frequency was 0.78 +/- 0.04 Hz in left ventricular myocardium and 2.80 +/- 0.31 Hz in right atrial myocardium (p < 0.01) at 27 degrees C. (2) The minimum stiffness frequency was 41% higher in non-failing compared to failing left ventricular human myocardium. (3) Over a wide range of experimental temperatures, the minimum stiffness frequency changed, with a Q10 of at least 2.7. (4) beta-Adrenergic stimulation significantly (p < 0.05) increased the minimum stiffness to 18 +/- 3% higher frequencies and significantly (p < 0.05) lowered contracture tension by 7 +/- 1%. CONCLUSIONS: The contractility of human heart muscle is not only regulated by excitation-contraction coupling but also by modulation of intrinsic properties of the actomyosin system. Acute and chronic alterations of cross-bridge kinetics have been demonstrated, which play a significant role in the physiology and pathophysiology of the human heart. (+info)
A single conductance pore for chloride ions formed by two cystic fibrosis transmembrane conductance regulator molecules.
(24/7624)
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase (PKA)- and ATP-regulated chloride channel, whose gating process involves intra- or intermolecular interactions among the cytosolic domains of the CFTR protein. Tandem linkage of two CFTR molecules produces a functional chloride channel with properties that are similar to those of the native CFTR channel, including trafficking to the plasma membrane, ATP- and PKA-dependent gating, and a unitary conductance of 8 picosiemens (pS). A heterodimer, consisting of a wild type and a mutant CFTR, also forms an 8-pS chloride channel with mixed gating properties of the wild type and mutant CFTR channels. The data suggest that two CFTR molecules interact together to form a single conductance pore for chloride ions. (+info)