Pathology of Naturally Occurring Bacillary Hemoglobinuria in Cattle.

Clostridium haemolyticum causes bacillary hemoglobinuria (BH), an infectious and usually fatal disease that occurs mostly in cattle, which is clinically characterized by jaundice, hemoglobinuria, and anemia. The trematode Fasciola hepatica has been commonly reported as the main predisposing factor that triggers this condition. The authors evaluated 20 naturally occurring cases of bovine BH to characterize the pathology and pathogenesis of the disease. Grossly, the most consistent finding was a large, frequently single focus of necrosis surrounded by a red to purple halo, observed most frequently on the parietal surface of the right and left hepatic lobes. Other findings were jaundice, dark-brown discoloration of kidneys, and red urine in the urinary bladder. Microscopically, characteristic lesions were locally extensive, necrotizing hepatitis with thrombosis and numerous intralesional Gram-positive rod-shaped bacteria, and acute renal tubular necrosis. By immunohistochemistry, many hepatocytes outside the necrotic focus in the liver were positive for activated caspase 3, suggesting that those cells were undergoing apoptosis. Ultrastructural evaluation revealed hepatocyte necrosis, hemolysis, and clumps of vegetative and sporulating bacilli within the liver. Polymerase chain reaction for the C. haemolyticum beta toxin gene was positive in randomly selected liver samples. No gross or microscopic lesions indicative of fascioliasis were detected in the liver of any animal, suggesting that other yet undetermined predisposing factors were associated with these cases of BH.

The relation between dicarbocyanine dye fluorescence and the membrane potential of human red blood cells set at varying Donnan equilibria.

The fluorescence, F, of two dicarbocyanine dyes, diS-C3(5) and diI-C3(5), depends both on the membrane potential, E, and on the intracellular pH, pHc, or human red blood cells. Compositions of isotonic media have been devised in which the equilibrium Donnan potential, E, varies at constant pHc and in which pHc varies at constant E. Dye fluorescence measurements in these suspensions yield calibrations of +1.7 % delta F/mV for diS-C3(5) and +0.6 % delta F/mV for diI-C3 (5). While pHo does not affect F of either dye, changes in pHc of 0.1 unit at constant E cause changes of F equivalent to those induced by 2--3mV. Based on these results, a method is given for estimating changes in E from dye fluorescence in experiments in which E and pHc co-vary. The relation of F to E also depends in a complex way on the type and concentration of cells and dye, and the wavelengths employed. The equilibrium calibration of dye fluorescence, when applied to diffusion potentials induced by 1 microM valinomycin, yields a value for the permeability ratio, PK.VAL/PCl, of 20 +/- 5, in agreement with previous estimates by other methods. The calibration of F is identical both for diffusion potentials and for equilibrium potentials, implying that diC-C3(5) responds to changes in voltage independently of ionic fluxes across the red cell membrane. Changes in the absorption spectra of dye in the presence of red cells in response to changes in E show that formation of nonfluorescent dimers contributes to fluorescence quenching of diS-C3(5). In contrast, only a hydrophobic interaction of dye monomers need be considered for diI-C3(5), indicating the occurrence of a simpler mechanism of fluorescence quenching.

Ionic and osmotic equilibria of human red blood cells treated with nystatin.

Human red blood cells have been incubated in the presence of nystatin, which allows Na and K, as well as Cl and pH to equilibrate rapidly when cell volume is set with external impermeant sucrose. The intracellular mean ionic activity coefficients, relative to values in the extracellular solution, for KCl and NaCl are 1.01 +/- 0.02 and 0.99 +/- 0.02 (SD, n = 10), respectively, and are independent of external pH, pH o, and of [sucrose]o. With nystatin the dependence of red cell volume on [sucrose]o deviates from ideal osmotic behavior by as much as a factor of three. A virial equation for the osmotic coefficient, phi, of human hemoglobin, Hb, accounts for the cell volumes, and is the same as that which describes Adair's measurements of phi Hb for Hb isolated from sheep and ox bloods. In the presence of nystatin the slope of the acid-base titration curve of the cells is independent of cell volume, implying that the charge on impermeant cellular solutes is independent of Hb concentration at constant pH. By modifying the Jacobs-stewart equations (1947. J. Cell. Comp. Physiol. 30: 79--103) with the osmotic coefficients of Hb and of salts, a nonideal thermodynamic model has been devised which predicts equilibrium Donnan ratios and red cell volume from the composition of the extracellular solution and from certain parameters of the cells. In addition to accounting for the dependence of cell volume on osmotic pressure, the model also describes accurately the dependence of Donnan ratios and cell volumes on pHo either in the presence or absence of nystatin.

Electrodiffusion, barrier, and gating analysis of DIDS-insensitive chloride conductance in human red blood cells treated with valinomycin or gramicidin.

Current-voltage curves for DIDS-insensitive Cl- conductance have been determined in human red blood cells from five donors. Currents were estimated from the rate of cell shrinkage using flow cytometry and differential laser light scattering. Membrane potentials were estimated from the extracellular pH of unbuffered suspensions using the proton ionophore FCCP. The width of the Gaussian distribution of cell volumes remained invariant during cell shrinkage, indicating a homogeneous C1- conductance among the cells. After pretreatment for 30 min with DIDS, net effluxes of K+ and Cl- were induced by valinomycin and were measured in the continued presence of DIDS; inhibition was maximal at approximately 65% above 1 microM DIDS at both 25 degrees C and 37 degrees C. The nonlinear current-voltage curves for DIDS-insensitive net Cl- effluxes, induced by valinomycin or gramicidin at varied [K+] o, were compared with predictions based on (1) the theory of electrodiffusion, (2) a single barrier model, (3) single occupancy, multiple barrier models, and (4) a voltage-gated mechanism. Electrodiffusion precisely describes the relationship between the measured transmembrane voltage and [K+]o. Under our experimental conditions (pH 7.5, 23 degrees C, 1-3 microM valinomycin or 60 ng/ml gramicidin, 1.2% hematocrit), the constant field permeability ratio PK/PCl is 74 +/- 9 with 10 microM DIDS, corresponding to 73% inhibition of PCl. Fitting the constant field current-voltage equation to the measured Cl- currents yields PCl = 0.13 h-1 with DIDS, compared to 0.49 h-1 without DIDS, in good agreement with most previous studies. The inward rectifying DIDS-insensitive Cl- current, however, is inconsistent with electrodiffusion and with certain single-occupancy multiple barrier models. The data are well described either by a single barrier located near the center of the transmembrane electric field, or, alternatively, by a voltage-gated channel mechanism according to which the maximal conductance is 0.055 +/- 0.005 S/g Hb, half the channels are open at -27 +/- 2 mV, and the equivalent gating charge is -1.2 +/- 0.3.

Voltage dependence of DIDS-insensitive chloride conductance in human red blood cells treated with valinomycin or gramicidin.

Net K and Cl effluxes induced by valinomycin or by gramicidin have been determined directly at varied external K, denoted by [K]o, in the presence and absence of the anion transport inhibitors DIDS (4,4'-diiso-thiocyano-2,2'-disulfonic acid stilbene), and its less potent analogue SITS (4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid). The results confirm that pretreatment with 10 microM DIDS, or 100 microM SITS, for 30 min at 23 degrees C inhibits conductive Cl efflux, measured in the continued presence of the inhibitors at 1 mM [K]o, by only 59-67%. This partial inhibition by 10 microM DIDS at 1 mM [K]o remains constant when the concentration of DIDS, or when the temperature or pH during pretreatment with DIDS, are increased. Observations of such partial inhibition previously prompted the postulation of two Cl conductance pathways in human red blood cells: a DIDS-sensitive pathway mediated by capnophorin (band 3 protein), and a DIDS-insensitive pathway. The present experiments demonstrate that at [K]o corresponding to values of EK between -35 and 0 mV the DIDS-insensitive component of net Cl efflux is negligible, being < or = 0.1 muMol/g Hb/min, both with valinomycin (1 microM) and with gramicidin (0.06 microgram/ml). At lower [K]o, where EK is below approximately -35 mV, the DIDS-insensitive fraction of net Cl efflux increases to 2.6 muMol/g Hb/min with valinomycin (1 microM), and to 4.8 muMol/g Hb/min with gramicidin (0.06 microgram/ml). With net fluxes determined from changes in mean cell volume, and with membrane potentials measured from changes in the external pH of unbuffered red cell suspensions, a current-voltage curve for DIDS-insensitive Cl conductance has been deduced. While specific effects of varied [K]o on net Cl efflux are unlikely but cannot strictly be ruled out, the results are consistent with the hypothesis that DIDS-insensitive Cl conductance turns on at an Em of approximately -40 mV.

Proton (or hydroxide) fluxes and the biphasic osmotic response of human red blood cells.

Upon exposure of human red blood cells to hypertonic sucrose, the fluorescence of the potentiometric indicator 3,3'-dipropylthiadicarbocyanine iodide, denoted diS-C3(5), displays a biphasic time course indicating the rapid development of an inside-positive transmembrane voltage, followed by a slow DIDS (4,4'-diisothiocyano-2,2'-disulfonic acid stilbene)-sensitive decline of the voltage. In addition to monitoring membrane potential, proton (or hydroxide) fluxes were measured by a pH stat method, cell volume was monitored by light scattering, and cell electrolytes were measured directly when red cells were shrunken either with hypertonic NaCl or sucrose. Shrinkage by sucrose induced an initial proton efflux (or OH- influx) of 5.5 mu eq/g Hb.min and a Cl shift of 21-31 mu eq/g Hb in 15 min. Upon shrinkage with hypertonic NaCl, the cells are initially close to Donnan equilibrium and exhibit no detectable shift of Cl or protons. Experiments with the carbonic anhydrase inhibitor ethoxzolamide demonstrate that for red cell suspensions exposed to air and shrunken with sucrose, proton fluxes mediated by the Jacobs-Stewart cycle contribute to dissipation of the increased outward Cl concentration gradient. With maximally inhibitory concentrations of ethoxzolamide, a residual proton efflux of 2 mu eq/g Hb.min is insensitive to manipulation of the membrane potential with valinomycin, but is completely inhibited by DIDS. The ethoxzolamide-insensitive apparent proton efflux may be driven against the electrochemical gradient, and is thus consistent with HCl cotransport (or Cl/OH exchange). The data are consistent with predictions of equations describing nonideal osmotic and ionic equilibria of human red blood cells. Thus osmotic equilibration after shrinkage of human red blood cells by hypertonic sucrose occurs in two time-resolved steps: rapid equilibration of water followed by slower equilibration of chloride and protons (or hydroxide). Under our experimental conditions, about two-thirds of the osmotically induced apparent proton efflux is mediated by the Jacobs-Stewart cycle, with the remainder being consistent with mediation via DIDS-sensitive HCl cotransport (or Cl/OH exchange).

Lack of erythrocyte superoxide dismutase change during human senescence.

Superoxide dismutase (SOD), widely distributed in aerobic organisms, catalyzes dismutation of the superoxide free radical, O2-, to oxygen and hydrogen peroxide and apparently protects against oxygen toxicity. In human erythrocytes, O2- arises from autoxidation of oxyhemoglobin and SOD activity is copper-dependent. Since human erythrocyte copper content has been reported to decline in the fifth decade of life, we investigated the age dependence of human erythrocyte SOD activity. The mean SOD activity, assayed by inhibition of epinephrine autoxidation, was 415 +/- 66 units/g cells or 50 +/- 11 units/mg non-Hb protein. No significant difference was observed between young and old adults, and no correlations were detected with sex, state of health of the donor, or with blood hemoglobin content. The lack of general decline of SOD activity with age narrows the possible mechanisms for an involvement of O2- in senescence. SOD may yet decline in other longer-lived tissues or, as suggested by Fridovich, a constant low level of damage may be caused by imperfect scavenging of O2- by SOD. If such a mechanism is operative, it appears not to affect synthesis of SOD in erythrocyte precursor cells into the eighth decade of human life.

Segmental pulmonary vascular responses to ATP in rat lungs: role of nitric oxide.

ATP exhibits vascular pressor and depressor responses in a dose- and tone-dependent manner. The vascular site of ATP-induced contraction or dilation has not previously been characterized. Using the vascular occlusion technique, we investigated the effects of ATP in isolated rat lungs perfused with autologous blood (hematocrit = 20%) and described its action during resting and elevated tone in terms of changes in resistances of the small and large arteries and veins. During resting tone, ATP (10(-5) M) caused contraction primarily in the small arteries and, to some extent, in the small veins, suggesting that P2x purinoceptors are present in these small vessels. During hypoxia, ATP caused dilation primarily in the small arteries, suggesting that P2y purinoceptors are predominant in small arteries. During U-46619-induced contraction, which occurred evenly throughout the four segments, ATP caused dilation in the large arteries and veins but not in the small arteries and veins. After treatment with N omega-nitro-L-arginine to inhibit nitric oxide synthesis, ATP-induced contraction was potentiated, and its dilatory effects during hypoxia were attenuated. The action of ATP was independent of prostanoids, because its constrictor and dilatory responses were not affected significantly by indomethacin. In conclusion, the results indicate that the effects of ATP on the pulmonary vasculature are primarily due to P2x and P2y purinoceptors in the small arteries. Contribution of these purinoceptors in other vessels to changes in total vascular resistance in rat lung was minor.

Cellular deformability of normoxic and hypoxic mammalian red blood cells.

The deformability of red blood cells is important in the microcirculation where capillary diameters are often smaller than those of the red blood cells. In the present study, ektacytometry was used to examine the effect of hypoxia on the deformability of red blood cells from five mammalian species: Human, cat, rat, rabbit, and dog. Deformability was characterized in both normoxic (PO2 = 129 +/- 6 mm Hg) and hypoxic (PO2 = 47 +/- 6 mm Hg) conditions in two different ways. First, we used the Elongation Index (EI) which quantitates the extent of elongation of red blood cells in response to increasing fluid shear stress; second, we used the Elongation Constant (EC), which quantitates the exponential dependence of the fraction of maximal elongation on the varying shear stress. The EI was measured at high shear stresses (150-250 dyn/cm2), as well as at lower shear stresses (15, 32 and 64 dyn/cm2) that occur in the microcirculation. In response to hypoxia at high shear stresses, the EI of the rat red blood cells decreased by 9.3% (P < 0.05), but was not altered in the other four species studied. Moreover, in all five species, the EC and EI at the lower shear stresses were unaltered in response to hypoxia. These ektacytometry experiments indicate that (1) the elongation constant is a new and useful parameter for characterizing the deformability of red blood cells and (2), the deformability of human, cat, dog, and rabbit red blood cells is unaltered by hypoxia. The results constrain the possible mechanisms that could account for the observation that hypoxia decreases the filterability of certain species of red blood cells, which was reported previously.

Partial requirements for in vitro survival of human red blood cells.

Some of the requirements for survival of human red blood cells were studied in vitro at 25 and 37 degrees C for 1--2 weeks. During the first week at 25 degrees C in Krebs-Ringer bicarbonate medium with glucose, the cells at 2--5% hematocrit (HCT) maintained normal K+, Na+, and water contents with negligible hemolysis. After six days ion gradients decreased, preceded by decline of ATP. With adenosine, ATP was maintained for 1--2 weeks. Sustained in vitro survival of human red blood cells at 25 or 37 degrees C requires constant pHo and sufficient substrates to support a glycolytic carbon flux as well as a nitrogen flux via nucleotide turnover. In Earle's salts buffered with HEPES and supplemented with glucose, Eagle's essential vitamins, albumin, and antibiotics, suspensions at 0.1% HCT exhibited constant pH at 7.39 +/- 0.03 for at least two weeks at 37 degrees C. With glucose alone, ATP declined steadily to negligible levels despite constant pHo, but 0.1 mM adenine supported ATP for one week. Also, several amino acids partially prevented the decline of reduced glutathione during the first week at 37 degrees C. These results and current knowledge of red cell metabolism suggest a new defined medium for experiments requiring long term incubations, and extend the characterization of human red cell in vitro survival to a time period not previously studied.

Membrane potentials associated with Ca-induced K conductance in human red blood cells: studies with a fluorescent oxonol dye, WW 781.

A divalent anionic dye, bis-[3-methyl-1-p-sulfophenyl-5-pyrazolone-(4)]-pentamethine oxonol (WW 781) is a rapidly responding fluorescent indicator of KCl diffusion potentials induced in human red blood cells with valinomycin, gramicidin, and with the Ca ionophore A 23187 in the presence of external Ca. WW 781 has a sensitivity of 0.13% delta F/mV, a detection limit of 10 mV, a response time of less than 1 sec, and exhibits a decrease in fluorescence intensity upon hyperpolarization without detectable shifts in absorption or emission peaks. This dye does not perturb the normal resting potential, and unlike the slow permeant cyanine dyes, does not inhibit Ca-induced K conductance in human red blood cells. However, WW 781 does stimulate Ca-induced unidirectional Rb efflux. With Ca plus A 23187, the initial rapid change in dye fluorescence is sensitive to [Ca]o and to [A 23187], is reversible with excess EGTA, and is inhibited by quinine, oligomycin, and by trifluoperazine. A biphasic dependence of hyperpolarization on Ko is evident at pH 6, where the ionic selectivity of activation is K, Rb greater than Cs greater than Na and that of conductance is K, Rb greater than Cs. Conditions were defined which permitted continuous monitoring of Em for at least 10 min, and the time dependence of the Ca-induced potentials was characterized. Since the properties of the Ca-induced changes in dye fluorescence correlate well with the known characteristics of Ca-induced K permeability, we conclude that WW 781 is a useful indicator of changes in Em, provided that sufficient controls are employed to separate direct effects of Ca on dye fluorescence from the effects of Em on fluorescence.

Relationship between the shape and the membrane potential of human red blood cells.

Microscopic observations of isotonic suspensions of human red blood cells demonstrate that cell shape is unaltered when the transmembrane electrical potential, or Em, is set in the range -85 to + 10 mV with valinomycin at varied external K+, or Ko X Em was measured with the fluorescent potentiometric indicator, diS-C3(5), as calibrated by a delta pH method. Repeating Glaser's experiments in which echinocytosis was attributed to hyperpolarization, we found that at low ionic strength the pH-dependent effects of amphotericin B appear to be unrelated to Em. The effects of increased intracellular Ca2+, or Cac, on echinocytosis and on Em are separable. With Ca ionophore A23187 half-maximal echinocytosis occurs at greater Cao than that which induces the half-maximal hyperpolarization associated with Ca-induced K+ conductance (Gardos effect). Thus, cells hyperpolarized by increased Cac remain discoidal when Ca is below the threshold for echinocytosis. With A23187 and higher Cao, extensive echinocytosis occurs in cells which are either hyperpolarized or at their resting potential. The Ca-activation curve for echinocytosis is left-shifted by low Ko, a new observation consistent with increased DIDS-sensitive uptake of 45Ca by hyperpolarized cells. These results support the following conclusions: (1) the shape and membrane potential of human red blood cells are independent under the conditions studied; (2) in cells treated with A23187, the Gardos effect facilitates echinocytosis by increasing Cac.

Calcium, cell shrinkage, and prolytic state of human red blood cells.

The effects of intracellular calcium, or Cac, on the Na permeability of human red blood cells were examined during 3-h incubations with the Ca ionophore A23187 and varied external Ca, Cao. Above 3 microM Cao, Nac increased significantly as ATP decreased. Maintenance of normal ATP with vanadate did not prevent the gain of Nac. Similar amounts of Nac were gained in 3 h by ouabain-treated cells exposed to the K ionophore valinomycin or by cells osmotically shrunken. Cells shrunken with sucrose also exhibited partial loss of Kc. When the cells with increased Nac were subsequently transferred to Na-free, high-K medium, the Nac and Kc that had changed slowly over 3 h returned toward normal within 10 min. The development of irreversible high cation permeability in shrunken cells was not prevented by a variety of transport inhibitors. These observations and cell volume distributions suggest that prolonged shrinkage induces a subpopulation of cells to become highly cation permeable, or "prolytic". The major effect of Cac on Na permeability appears to be an indirect consequence of cell shrinkage due to KCl loss.

Membrane lipid fluidity and filterability of red blood cells from adults and newborns.

Membrane lipid fluidity was reexamined in red blood cells and ghosts from adults and newborns. Fluorescence anisotropies of the hydrophobic probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and perylene were significantly and substantially greater in fresh intact red cells from newborns than from adults; however, no significant difference was detected with the polar fluorophores, 12-(9-anthroyl) stearic acid and retinol. These results suggest that probes in the hydrophobic core of the membrane have less motional freedom in red cells from newborns than from adults, whereas probe motional freedom in the polar lipid headgroup regions of the membranes is similar for both cell types. DPH fluorescence anisotropy increased upon making white ghosts or upon storage of blood. Temperature studies indicated that DPH fluorescence anisotropy in fresh intact neonatal red cells is increased by an amount corresponding to that produced by cooling adult red cells by 22 degrees C. Elevated intracellular calcium decreased red cell filterability without affecting DPH fluorescence anisotropy of ghost membranes. This result suggests that the effect of calcium in reducing filterability is independent of alterations in membrane lipid motional freedom. It is unlikely that the decreased lipid motional freedom of red cells from newborns contributes significantly to their decreased filterability.