1. Intracellular recordings were obtained from supraoptic magnocellular neurosecretory cells (MNCs) in superfused explants of rat hypothalamus. Application of ATP and UTP, but not adenosine, produced TTX-insensitive depolarizations accompanied by increases of input conductance.2. The P2X agonists ,,B-methylene ATP, l,y-methylene ATP and 2-methylthio ATP mimicked the effects of ATP in >77 % of the cells tested. Depolarizing responses to ATP were reversibly inhibited by PPADS (pyridoxal-phosphate-6-azophenyl-2', 4'-disulphonic acid; IC50 ; 0 5 uM), a selective P2X antagonist.3. The reversal potential of responses to ATP (-37 mV) was not strongly affected by intracellular Cl-injection or by removal of Cl-from the external solution. The reversal potential of responses to the most potent P2X agonist, a,f-methylene ATP, was -29 mV. These values suggest the involvement of non-selective cationic channels, a finding which is consistent with the ionotropic cationic channel structure of cloned P2X purinoceptors.4. The reversal potential of UTP-mediated responses (-33 mV) was also consistent with the involvement of non-selective cationic channels. Since cloned P2u receptors display homology with G-protein-coupled receptors, cationic channels modulated by UTP are probably different from those mediating P2X responses.
Sickle cell anemia is a disease of abnormal rheology caused by acute and reversible, as well as chronic and irreversible, changes in the properties and deformability of sickle erythrocytes. Deformability is determined by several factors, including intracellular sickle hemoglobin polymerization, the abnormal membrane properties of sickle cells, and the abnormal rheological properties of the soluble concentrated hemoglobin solution within dense sickle red blood cells. In this study, we used a 5-microns pore nickel mesh filter to evaluate quantitatively the effects of these factors on the filterability of erythrocytes containing sickle hemoglobin. We used sickle trait and sickle/beta(+)-thalassemia cells, because they have minimal membrane abnormalities or density heterogeneity, to investigate the effects of polymer formation on rheological properties. We found that filterability of these cells is sensitive to small amounts of intracellular polymer and that impaired filtration is linearly related to oxygen-dependent polymer formation, up to a polymer fraction of 0.3. By increasing the proportion of dense cells in populations of normal cells or cells from individuals with sickle syndromes and equilibrating these cells with gas ligands, we estimate that polymerization, even at 95% saturation, contributes twice as much to impaired filterability of sickle erythrocytes as the abnormal membranes in homozygous sickle cell disease. At lower saturation values, the effects of polymer are even greater. The viscosity of the concentrated hemoglobin in dense cells had the smallest effect, over physiologically relevant saturation values. These results emphasize the importance of sickle hemoglobin polymerization in the pathogenesis of sickle cell disease and should help define its pathophysiology and responses to therapy in quantitative terms.
Sickle cell disease pathophysiology is mediated by acute and chronic impairment of cell flexibility due to the formation of intracellular sickle hemoglobin (Hb S) polymer as cells are partially deoxygenated in the microcirculation. We have recently developed a method to measure the relationship between the formation of intracellular polymerized Hb S and cell filtration. In this study, we have used this method to examine whether sickle cell morphology, independent of Hb S polymer fraction, had an effect on cell rheology. We primarily use sickle trait (AS) and Hb S-beta(+)-thalassemia (S-beta(+)-thal) erythrocytes with low hemoglobin F levels, which have normal membranes and few or no dense cells, to remove these confounding effects. We find that the relationship between filtration and the percentages of each "type" of morphological deformation of AS erythrocytes was different from that of the S-beta(+)-thal erythrocytes. In addition, we find that while the filtration of AS erythrocytes as a function of oxygen saturation was similar, whether measured during deoxygenation or reoxygenation, the relationship between the percentages of each type of deformed erythrocyte and oxygen saturation demonstrated hysteresis during oxygenation-deoxygenation experiments. Transmission electron microscopy, for both elongated and irregularly shaped cells, showed that similarly distorted cells could have very different amounts and alignment of polymer. These results suggests that cell morphology per se is not strongly related to filtration, whereas calculated intracellular Hb S polymer fraction predicts loss of filtration of AS and S-beta(+)-thal erythrocytes well. Measured or calculated polymer fraction values would appear to be a better parameter for the study of sickle cell disease pathophysiology and response to treatment than cell morphology studies.
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