Péter Arányi

Kinetics of the hormone-receptor interaction. Competition experiments with slowly equilibrating ligands.

Kinetics of formation of protein-tracer complex in the presence of competitor were calculated. The parameters were chosen so that they should realistically describe the in vitro association of steroid hormones with their receptors. Time necessary for equilibration depends on four rate constants in addition to initial concentrations and may be more than 1000 min. Competitors forming complexes with the protein that dissociate faster than the protein tracer complex have relative binding affinities apparently decreasing with incubation time. Conversely, relative binding affinity apparently increases with time if the protein-competitor complex dissociates more slowly than the protein-tracer complex. Moreover, lack of equilibration is not easily detected. It is suggested that kinetic analyses, more detailed than usual, precede competition experiments.

Postconditioning of the lower limb--protection against the reperfusion syndrome.

BACKGROUND Postconditioning-alternating brief cycles of reperfusion/reocclusion applied at the beginning of revascularization-is a potent therapeutic technique, attenuating ischemia-reperfusion injury. Vascular surgery on the lower limb with ischemia-reperfusion injury may give rise to serious systemic complications [organ dysfunction syndrome (MODS), systemic inflammatory response syndrome (SIRS)], a phenomenon called reperfusion-syndrome. MATERIAL AND METHODS We studied the effects of postconditioning on reperfusion-syndrome in a rodent experimental model. Wistar rats underwent 180 min of bilateral lower limb ischemia using an infrarenal crossclamping of the abdominal aorta. Postconditioning consisted of six cycles of 10-s aortic occlusion/10-s declamping at the beginning of reperfusion. Microcirculation of the lower limb was detected with laser Doppler flowmeter. After 4 h of reperfusion, plasma, urine, and histologic samples were collected. RESULTS One hundred eighty-minute ischemia resulted in significant hemodynamic changes after reperfusion. Postconditioning affected the character of the microcirculatory flow, the limb circulation stabilized with hyperemia during reperfusion. Postconditioning caused a significant reduction in systemic inflammatory response (TNF-α, oxygen-derived free radicals). The laboratory and histologic samples implied a significant decrease in distant organ (lung and renal) dysfunctions after postconditioning. CONCLUSION Postconditioning proves to be capable of conferring protection against different organ injuries caused by longer circulatory occlusions during elective major vascular operations.

Effect of calcium on chick thymus glucocorticoid receptor

Abstract The effect of Ca 2+ in the millimolar range was investigated on the heat stability of chick thymus receptor in the absence of glucocorticoids. Size and DNA-binding of glucocorticoid-receptor complex was also studied after calcium-treatment. Ca 2+ reduced dramatically and in a concentration-dependent way the heat stability of the receptor at 25°C in the absence of hormone. Binding of triamcinolone acetonide-receptor complex to DNA-cellulose was also strongly decreased when 0.5–8mM Ca 2+ was present during activation of the complex or if cytosol was pre-treated with Ca 2+ before addition of the steroid. Ca 2+ treatment caused a significant reduction in the size of receptor-hormone complex as determined by Sephacryl S-200 chromatography. The results suggest that Ca 2+ may have an in vivo regulatory role decreasing or inhibiting the expression of glucocorticoid hormone action under specific conditions.

Determine rate constants of interaction of steroid receptors with non-labelled ligands

Abstract Theoretical analysis of the time course of receptor-ligand association and dissociation in the presence of a competing ligand revealed a method that rate constants of receptor ligand interaction could be determined for non-labelled ligands. This requires a short term competition measurement for determining association constants, and an exchange assay for determining dissociation rate constants. Theory has been verified by comparison of rate constants of glucocorticoid receptor-steroid interactions determined the above ways with those determined by use of radiolabelled ligands. The novel methods developed here would permit determination of rate constants for a number of processes that were unavailable to study because of lack of radiolabelled ligand. Moreover, the exchange method of determination of dissociation rate constant is uniquely suitable in case of very slowly dissociating ligand, even if the latter is available in radiolabelled form.

Kinetics of the glucocorticoid hormone-receptor interaction: False association constant determined in slowly equilibrating systems

The usual way of in vitro determination of association constants for hormone-receptor complexes is criticized. It is shown that if incubation time is short, relative to the half-life of the hormone-receptor complex, the value of the apparent Ka is proportional to the time of incubation. No sign of lack of equilibrium is apparent from the Scatchard plots. The case of rapidly denaturing receptor molecule is also discussed, with similar conclusions. Although terminology and examples are taken from the field of the glucocorticoid receptor research, all deductions are valid for other systems with similar association (and denaturation or monomolecular transformation) mechanisms and kinetic parameters.

Comparative study of glucocorticoid sensitivity and receptors in lymphoid tissues.

Abstract Glucocorticoid sensitivity of different lymphoid tissues of avian and mammalian origin was investigated. As a measure of glucocorticoid effect variation of thymidine kinase activity was selected. By this method an order of sensitivity was established among various species and tissues. Dexamethasone uptake by isolated lymphocytes and specific binding to cytosol receptors was also examined. No direct correlation was found between glucocorticoid sensitivity and either binding capacity (receptor number per cell) or affinity of receptor for hormone. The presence of one homogeneous binding site was detected in each tissue examined both by isolated cell technique and cytosol saturation analysis in the range of 1–40 nM of [3H]-dexamethasone concentration. In all cases about 80% of the bound dexamethasone was associated with cell nucleus in our system. Dexamethasone binding of the hydrocortisone resistant subpopulation of chicken lymphoid tissues was studied after in vivo treatment with high doses of hydrocortisone. The surviving cells contained an amount of dexamethasone receptor cell similar to that of the whole population. A theoretical analysis of the steady-state receptor cycle gives an explanation for the differences between the values of KA determined in intact cells or in cellfree systems.

Attenuation of Skeletal Muscle and Renal Injury to the Lower Limb following Ischemia-Reperfusion Using mPTP Inhibitor NIM-811

Introduction Operation on the infrarenal aorta and large arteries of the lower extremities may cause rhabdomyolysis of the skeletal muscle, which in turn may induce remote kidney injury. NIM-811 (N-metyl-4-isoleucine-cyclosporine) is a mitochondria specific drug, which can prevent ischemic-reperfusion (IR) injury, by inhibiting mitochondrial permeability transition pores (mPTP). Objectives Our aim was to reduce damages in the skeletal muscle and the kidney after IR of the lower limb with NIM-811. Materials and methods Wistar rats underwent 180 minutes of bilateral lower limb ischemia and 240 minutes of reperfusion. Four animal groups were formed called Sham (receiving vehicle and sham surgery), NIM-Sham (receiving NIM-811 and sham surgery), IR (receiving vehicle and surgery), and NIM-IR (receiving NIM-811 and surgery). Serum, urine and histological samples were taken at the end of reperfusion. NADH-tetrazolium staining, muscle Wet/Dry (W/D) ratio calculations, laser Doppler-flowmetry (LDF) and mean arterial pressure (MAP) monitoring were performed. Renal peroxynitrite concentration, serum TNF-α and IL-6 levels were measured. Results Less significant histopathological changes were observable in the NIM-IR group as compared with the IR group. Serum K+ and necroenzyme levels were significantly lower in the NIM-IR group than in the IR group (LDH: p<0.001; CK: p<0.001; K+: p = 0.017). Muscle mitochondrial viability proved to be significantly higher (p = 0.001) and renal function parameters were significantly better (creatinine: p = 0.016; FENa: p<0.001) in the NIM-IR group in comparison to the IR group. Serum TNF-α and IL-6 levels were significantly lower (TNF-α: p = 0.003, IL-6: p = 0.040) as well as W/D ratio and peroxynitrite concentration were significantly lower (p = 0.014; p<0.001) in the NIM-IR group than in the IR group. Conclusion NIM-811 could have the potential of reducing rhabdomyolysis and impairment of the kidney after lower limb IR injury.

Muscle Fiber Viability, a Novel Method for the Fast Detection of Ischemic Muscle Injury in Rats

Acute lower extremity ischemia is a limb- and life-threatening clinical problem. Rapid detection of the degree of injury is crucial, however at present there are no exact diagnostic tests available to achieve this purpose. Our goal was to examine a novel technique - which has the potential to accurately assess the degree of ischemic muscle injury within a short period of time - in a clinically relevant rodent model. Male Wistar rats were exposed to 4, 6, 8 and 9 hours of bilateral lower limb ischemia induced by the occlusion of the infrarenal aorta. Additional animals underwent 8 and 9 hours of ischemia followed by 2 hours of reperfusion to examine the effects of revascularization. Muscle samples were collected from the left anterior tibial muscle for viability assessment. The degree of muscle damage (muscle fiber viability) was assessed by morphometric evaluation of NADH-tetrazolium reductase reaction on frozen sections. Right hind limbs were perfusion-fixed with paraformaldehyde and glutaraldehyde for light and electron microscopic examinations. Muscle fiber viability decreased progressively over the time of ischemia, with significant differences found between the consecutive times. High correlation was detected between the length of ischemia and the values of muscle fiber viability. After reperfusion, viability showed significant reduction in the 8-hour-ischemia and 2-hour-reperfusion group compared to the 8-hour-ischemia-only group, and decreased further after 9 hours of ischemia and 2 hours of reperfusion. Light- and electron microscopic findings correlated strongly with the values of muscle fiber viability: lesser viability values represented higher degree of ultrastructural injury while similar viability results corresponded to similar morphological injury. Muscle fiber viability was capable of accurately determining the degree of muscle injury in our rat model. Our method might therefore be useful in clinical settings in the diagnostics of acute ischemic muscle injury.

Effect of cross-linking on glucocorticoid receptor activation

Chick thymus cytosol glucocorticoid receptor complexed with [3H]-triamcinolone acetonide was heat-activated after treatment with diimidates of varying chain length. Diimidates with maximal effective reagent length shorter than 0.5 nm influenced DNA-cellulose binding only slightly, whereas sebacic diimidate (maximal effective reagent length 1.21 nm) blocked activation completely. Coupled activation - deactivation process induced in the presence of calcium was again inhibited only by the longer diimidates. Sebacic diimidate treated complex had a molecular mass similar to that of the molybdate stabilized nonactivated complex, as judged by gel permeation chromatography. It is suggested that prevention of size reduction by cross-linking blocks receptor activation as well.

The interaction of thrombin and heparin. Heat inactivation kinetics

Abstract Kinetic analysis of heat inactivation of thrombin in the presence of varying concentrations of heparin suggests that two forms of thrombin exist with respect to heparin sensitivity. One of them is inactivated by heat easily and cannot be stabilized with heparin. The other one interacts with heparin and is protected against heat inactivation at 54°C.