Russell C. Scaduto

Measurement of Mitochondrial Membrane Potential Using Fluorescent Rhodamine Derivatives

We investigated the use of rhodamine 123 (R123), tetramethylrhodamine methyl ester (TMRM), and tetramethylrhodamine ethyl ester (TMRE) as fluorescent probes to monitor the membrane potential of mitochondria. These indicator dyes are lipophilic cations accumulated by mitochondria in proportion to DeltaPsi. Upon accumulation, all three dyes exhibit a red shift in both their absorption and fluorescence emission spectra. The fluorescence intensity is quenched when the dyes are accumulated by mitochondria. These properties have been used to develop a method to dynamically monitor DeltaPsi of isolated rat heart mitochondria using a ratio fluorescence approach. All three dyes bound to the inner and outer aspects of the inner mitochondrial membrane and, as a result, were accumulated by mitochondria in a greater quantity than predicted by the Nernst equation. Binding to mitochondria was temperature-dependent and the degree of binding was in the order of TMRE > R123 > TMRM. The internal and external partition coefficients for binding were determined to correct for binding in the calculation of DeltaPsi. All three dyes suppressed mitochondrial respiratory control to some extent. Inhibition of respiration was greatest with TMRE, followed by R123 and TMRM. When used at low concentrations, TMRM did not suppress respiration. The use of these dyes and ratio fluorescence techniques affords a simple method for measurement of DeltaPsi of isolated mitochondria. We also applied this approach to the isolated perfused heart to determine whether DeltaPsi could be monitored in an intact tissue. Wavelength scanning of the surface fluorescence of the heart under various conditions after accumulation of TMRM indicated that the mitochondrial matrix-induced wavelength shift of TMRM also occurs in the heart cytosol, eliminating the use of this approach in the intact heart.

Hydrocortisone decreases retinal endothelial cell water and solute flux coincident with increased content and decreased phosphorylation of occludin

Corticosteroids provide an effective treatment to reduce edema for conditions in which the blood–brain or blood–retinal barrier is compromised. However, little is known about the mechanism by which these hormones affect endothelial cell function. We hypothesized that hydrocortisone would reduce transport of water and solutes across bovine retinal endothelial cell (BREC) monolayers coincident with changes to the tight junction protein occludin. Treatment of BREC with 103 nm hydrocortisone for two days significantly decreased water and solute transport across cell monolayers. Immunoblot analysis of occludin extracted in SDS or urea based buffers revealed a 1.65‐ or 2.57‐fold increase in content, respectively. A similar two‐fold increase in occludin mRNA was observed by real‐time PCR. Immunocytochemistry revealed hydrocortisone dramatically increased both occludin and ZO‐1 staining at the cell border. Additionally, 4 h of hydrocortisone treatment significantly reduced occludin phosphorylation. To our knowledge, this is the first example of a regulated decrease in occludin phosphorylation associated with increased barrier properties. In conclusion, hydrocortisone directly affects retinal endothelial cell barrier properties coincident with changes in occludin content, phosphorylation and tight junction assembly. Localized hydrocortisone therapy may be developed as a treatment option for patients suffering from retinal edema due to diabetes.

IGF-I prevents glutamate-mediated bax translocation and cytochrome C release in O4+ oligodendrocyte progenitors.

Oligodendroglial death due to overactivation of the AMPA/kainate glutamate receptors is implicated in white matter damage in multiple CNS disorders. We previously demonstrated that glutamate induces caspase‐3 activation and death of the late oligodendrocyte progenitor known as the pro‐oligodendroblast (pro‐OL) via activation of the AMPA/kainate glutamate receptors. We also demonstrated that IGF‐I had the unique ability to sustain activation of Akt in the pro‐OL and provide long‐term protection of these cells from glutamate‐mediated apoptosis. The goal of these studies was to investigate the mechanisms of glutamate toxicity and IGF‐I‐mediated survival in the pro‐OL. IGF‐I prevented glutamate‐induced loss of mitochondrial membrane potential, cytochrome c release, and caspase‐9 activation. In contrast to IGF‐I mediated survival mechanisms in neurons, IGF‐I had no effect on the influx or recovery of intracellular calcium levels or on levels of major pro‐ and anti‐apoptotic molecules including Bax or Bcl‐xL. Rather, IGF‐I prevented the glutamate‐induced translocation of Bax to the mitochondria. Moreover, IGF‐I prevented caspase‐3 activation in pro‐OLs as long as 8 h after exposure of the cells to glutamate, suggesting that delayed activation of IGF‐I‐mediated survival pathways can block glutamate‐mediated apoptosis in pro‐OLs. The results of these experiments define the mechanisms by which glutamate kills oligodendrocyte progenitor cells and by which IGF‐I blocks glutamate‐induced apoptosis in these cells. The data also demonstrate that IGF‐I disrupts the glutamate‐mediated apoptotic pathway in the pro‐OL through mechanisms that are distinct from its survival‐promoting actions in neurons.

Erythropoietin stimulates a rise in intracellular free calcium concentration in single early human erythroid precursors.

Erythropoietin and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulate the differentiation and proliferation of erythroid cells. To determine the cellular mechanism of action of these growth factors, we measured changes in intracellular free calcium concentration [( Cac]) in single human erythroid precursors in response to recombinant erythropoietin and GM-CSF. [Cac] in immature erythroblasts derived from cultured human cord blood erythroid progenitors was measured with fluorescence microscopy digital video imaging. When stimulated with erythropoietin, [Cac] in the majority of erythroblasts increased within 3 min, peaked at 5 min, and returned toward baseline at 10 min. The percentage of cells that responded to erythropoietin stimulation increased in a dose-dependent manner. Additional stimulation with GM-CSF in cells previously exposed to erythropoietin resulted in a second [Cac] increase. Immature erythroblasts treated with GM-CSF followed by erythropoietin responded similarly to each factor with a rise in [Cac]. The source of transient calcium is intracellular since erythroblasts were incubated in medium devoid of extracellular calcium. Our observations suggest that changes in [Cac] may be an intracellular signal that mediates the proliferative/differentiating effect of hematopoietic growth factors.

Influence of calcium and iron on cell death and mitochondrial function in oxidatively stressed astrocytes.

Astrocytes protect neurons and oligodendrocytes by buffering ions, neurotransmitters, and providing metabolic support. However, astrocytes are also vulnerable to oxidative stress, which may affect their protective and supportive functions. This paper examines the influence of calcium and iron on astrocytes and determines if cell death could be mediated by mitochondrial dysfunction. We provide evidence that the events associated with peroxide‐induced death of astrocytes involves generation of superoxide at the site of mitochondria, loss of mitochondrial membrane potential, and depletion of ATP. These events are iron‐mediated, with iron loading exacerbating and iron chelation reducing oxidative stress. Iron chelation maintained the mitochondrial membrane potential, prevented peroxide‐induced elevations in superoxide levels, and preserved ATP levels. Although increased intracellular calcium occurred after oxidative stress to astrocytes, the calcium increase was not necessary for collapse of mitochondrial membrane potential. Indeed, when astrocytes were oxidatively stressed in the absence of extracellular calcium, cell death was enhanced, mitochondrial membrane potential collapsed at an earlier time point, and superoxide levels increased. Additionally, our data do not support opening of the mitochondrial permeability transition pore as part of the mechanism of peroxide‐induced oxidative stress of astrocytes. We conclude that the increase in intracellular calcium following peroxide exposure does not mediate astrocytic death and may even provide a protective function. Finally, the vulnerability of astrocytes and their mitochondria to oxidative stress correlates more closely with iron availability than with increased intracellular calcium. J. Neurosci. Res. 55:674–686, 1999. 

Three-dimensional intracellular calcium gradients in single human burst-forming units-erythroid-derived erythroblasts induced by erythropoietin.

We have previously shown that the intracellular free Ca2+ increase induced by erythropoietin is likely related to differentiation rather than proliferation in human BFU-E-derived erythroblasts (1989. Blood. 73:1188-1194). Since cell differentiation involves transcription of specific regions of the genome, and since nuclear endonucleases responsible for single strand DNA breaks observed in cells undergoing differentiation are Ca2+ dependent, we investigated whether the erythropoietin-induced calcium signal is transmitted from cytosol to nucleus in this study. To elucidate subcellular Ca2+ gradients, the technique of optical sectioning microscopy was used. After determining the empirical three-dimensional point spread function of the video imaging system, contaminating light signals from optical planes above and below the focal plane of interest were removed by deconvolution using the nearest neighboring approach. Processed images did not reveal any discernible subcellular Ca2+ gradients in unstimulated erythroblasts. By contrast, with erythropoietin stimulation, there was a two- to threefold higher Ca2+ concentration in the nucleus compared to the surrounding cytoplasm. We suggest that the rise in nuclear Ca2+ may activate Ca2(+)-dependent endonucleases and initiate differentiation. The approach described here offers the opportunity to follow subcellular Ca2+ changes in response to a wide range of stimuli, allowing new insights into the role of regional Ca2+ changes in regulation of cell function.

Glutathione catabolism by the ischemic rat kidney

The glutathione (GSH) content of rat kidney decreases after cessation of blood flow, falling to 40% of control levels 35 min after renal artery occlusion [R. C. Scaduto, Jr., V. H. Gattone II, L. W. Grotyohann, J. Wertz, and L. F. Martin. Am. J. Physiol. 255 (Renal Fluid Electrolyte Physiol. 24): F911-F921, 1988]. Renal GSH levels remained depressed for at least 2 h after resumption of blood flow. Because GSH functions in the removal of free radicals, and lipid peroxidation is a free radical-initiated process that occurs in the ischemic kidney, we investigated the fate of this GSH pool in the ischemic kidney. Using high-performance liquid chromatography to measure thiols, we found the loss of GSH to be associated with a stoichiometric accumulation of cysteine in the kidney. Moreover, preischemic labeling of the renal GSH pool with 35S led to accumulation of [35S]cysteine during ischemia that had the same specific activity as that of tissue GSH. Formation of cysteine during ischemia was suppressed in rats pretreated with acivicin, an inhibitor of gamma-glutamyltransferase (gamma-GT), although the degree of suppression was small in comparison to the extent of gamma-GT inhibition. During the initial 2 min of blood reflow after ischemia, tissue cysteine returned to control levels, and a transient increase in the cysteine content of renal venous blood was observed. After ischemia, renal GSH levels remained depressed, but postischemic GSH levels could be increased by administration of N-acetylcysteine during the ischemic period.(ABSTRACT TRUNCATED AT 250 WORDS)

Ion channels in human erythroblasts. Modulation by erythropoietin.

To investigate the mechanism of intracellular Ca2+ ([Cai]) increase in human burst-forming unit-erythroid-derived erythroblasts by erythropoietin, we measured [Cai] with digital video imaging, cellular phosphoinositides with high performance liquid chromatography, and plasma membrane potential and currents with whole cell patch clamp. Chelation of extracellular free Ca2+ abolished [Cai] increase induced by erythropoietin. In addition, the levels of inositol-1,4,5-trisphosphate did not increase in erythropoietin-treated erythroblasts. These results indicate that in erythropoietin-stimulated cells, Ca2+ influx rather than intracellular Ca2+ mobilization was responsible for [Cai] rise. Both Ni2+ and moderately high doses of nifedipine blocked [Cai] increase, suggesting involvement of ion channels. Resting membrane potential in human erythroblasts was -10.9 +/- 1.0 mV and was not affected by erythropoietin, suggesting erythropoietin modulated a voltage-independent ion channel permeable to Ca2+. No voltage-dependent ion channel but a Ca(2+)-activated K+ channel was detected in human erythroblasts. The magnitude of erythropoietin-induced [Cai] increase, however, was insufficient to open Ca(2+)-activated K+ channels. Our data suggest erythropoietin modulated a voltage-independent ion channel permeable to Ca2+, resulting in sustained increases in [Cai].

Elevation of Renal Glutathione Enhances Ischemic Injury

In a previous study, we tested the hypothesis that an elevated level of renal glutathione (GSH) would protect the kidney from ischemic injury. However, prior elevation of GSH with GSH monoethylester enhanced then injury induced by 35 min of ischemia and blood reflow [Scaduto RC Jr, Gattone VH, Grotyohann LW, et al; Effect of an altered glutathione content on renal ischemic injury. Am J Physiol 1988;255:F911-F921]. Additionally, GSH monoethylester produced morphologic alterations in the absence of ischemia. Thus the greater ischemic injury observed after GSH ester pretreatment could have been due to a synergistic effect between the events caused by ischemia and the pretreatment. The present study was conducted to evaluate the utility of elevating renal GSH levels by administration of GSH. Administration of GSH (1 mmol/kg body weight) caused a 3-fold elevation of renal GSH levels and a 6-fold elevation of renal cysteine levels after 60 min without causing changes in renal morphology or GFR. After 35 min of renal artery occlusion and 90 min of blood reflow, animals pretreated with GSH had a much greater decline in GFR than untreated control animals. This enhancement of renal ischemic injury in GSH-treated animals was similar to that observed following administration of GSH monoethylester. We conclude that administration of GSH is the method of choice for elevation of renal GSH and that elevation of renal GSH leads to an enhanced ischemia-induced injury which is independent of the method employed to elevate renal GSH.

Effect of Mitochondrial Ca2+ on Hepatic Aspartate Formation and Gluconeogenic Flux

According to current evidence, the mechanism of action of many gluconeogenic hormones involves a rise in intracellular free Ca2+ (Charestet al., 1983; Sistareet al., 1985). There is general agreement that hepatic gluconeogenesis is controlled at several sites in the pathway by Ca2+ -and cAMP-mediated phosphorylations of cytosolic enzymes (Hers and Hue, 1983; Pilkiset al., 1986). Nevertheless, there is a perception that other Ca2+ -linked sites of action may be important; in particular, those which involve mitochondrial enzymes (Leverveet al., 1986; McCormack, 1985; Staddon and Hansford, 1987). The purpose of the study was to evaluate the significance of the stimulation by Ca2+ ofα-ketoglutarate dehydrogenase (McCormack, 1985) and pyruvate dehydrogenase (Oviasu and Whitton, 1984) which occurs following exposure of the liver to glucagon or phenylephrine. The dramatic decrease inα-toglutarate caused by these hormones (Siesset al., 1977) is rather convincingly due to the stimulation ofα-ketoglutarate dehydrogenase. Studies from this laboratory (LaNoueet al., 1983; Schoolwerth and LaNoue, 1983) showed thatα-ketoglutarate is a potent and physiologically important inhibitor of glutamate dehydrogenase. Stimulation ofα-ketoglutarate dehydrogenase relieves inhibition of glutamate dehydrogenase byα-ketoglutarate and, thereby, may stimulate gluconeogenesis from amino acids. Glucagon and phenylephrine are also known to stimulate gluconeogenesis from lactate (Hutsonet al., 1976; Kneeret al., 1979), and to stimulate alcohol oxidation (Ochs and Lardy, 1981) and the malate-aspartate cycle (Kneeret al., 1979; Leverveet al., 1986). These processes do not involve glutamate dehydrogenase but rather aspartate aminotransferase and the glutamate aspartate translocase (Rognstad and Katz, 1970; Williamsonet al., 1971).