Biomedical subjects

B Herman

Publications and source records attributed to B Herman.

At least 19 recordsLinked to original sources

PubMed1992

Platelet-derived growth factor (PDGF) alpha receptor activation modulates the calcium mobilizing activity of the PDGF beta receptor in Balb/c3T3 fibroblasts.

In order to determine whether distinct platelet-derived growth factor (PDGF) receptors (alpha and beta) can modulate the activity of one another, PDGF isoform (AA, BB, and AB)-stimulated changes in Ca2+i were monitored by digitized video microscopy in single cells upon sequential addition of PDGF isoforms. In Balb/c 3T3 fibroblasts, all PDGF isoforms were capable of stimulating increases in Ca2+i of 200-600% above basal levels, although with different potencies: BB greater than or equal to AB greater than AA. All cells were BB-PDGF-responsive, but only 74% of cells examined responded to AA-PDGF. The Ca2+i response elicited by BB-PDGF was inhibited by 60-75% in cells stimulated 10 min earlier with the AA isoform. The half-life of this inhibition was 22 min. In cells in which the alpha receptor was down-regulated by prolonged incubation with AA-PDGF, BB-induced Ca2+i responses were not inhibited. Pretreatment of cells with phorbol ester did not inhibit BB-PDGF-induced increases in Ca2+i, yet down-regulation of PKC activity prevented the AA-PDGF inhibition of BB-PDGF-induced Ca2+i responses. An increase in Ca2+i induced by AlF(4-)-stimulated IP3 generation did not inhibit a subsequent BB-PDGF Ca2+i response; however, attenuation of AA-PDGF-induced extracellular Ca2+ influx with EGTA prevented the inhibition of BB-PDGF-induced Ca2+i increases. Readdition of Ca2+ to the medium after removal of EGTA restored the inhibition of the BB-PDGF Ca2+i response. The inhibition of the BB-PDGF Ca2+i response by AA-PDGF was not caused by inhibition of PDGF receptor tyrosine autophosphorylation, which was unchanged after pretreatment with AA-PDGF. These results demonstrate: (a) that only a subpopulation of cells possess a functional alpha receptor-mediated response as assessed by AA-PDGF-induced increases in Ca2+i, whereas all cells possess the beta receptor-mediated responses; and (b) AA-PDGF and its associated alpha receptor can modulate the activity of the beta receptor through a mechanism that is dependent upon Ca(2+)-influx which may be controlled in part by PKC activation.

3T3 Cells↗

PubMed1992

A novel cytotoxicity screening assay using a multiwell fluorescence scanner.

A new assay using a multiwell fluorescence scanner was developed for screening cytotoxicity to cells cultured in 96-well microtiter plates. The assay is based on binding of propidium iodide to nuclei of cells whose plasma membranes have become permeable due to cell death. Fluorescence of propidium iodide measured with a multiwell fluorescence scanner increased in proportion to the number of permeabilized cells. After ATP depletion of hepatocytes and neonatal cardiac myocytes with metabolic inhibitors ("chemical hypoxia"), and exposure of Madine Darby canine kidney cells to the toxic chemical, HgCl2, propidium iodide fluorescence progressively increased. Increases of fluorescence were linearly proportional with release of lactate dehydrogenase into the culture medium. Employing this cytotoxicity screening assay, protection by various agents against lethal injury was evaluated in cultured hepatocytes during chemical hypoxia. Inhibitors of cysteine proteases (i.e., antipain, leupeptin, E-64), serine proteases (i.e., PMSF), and aspartic acid proteases (i.e., pepstatin A) did not protect against chemical hypoxia. In contrast, 1,10-phenanthroline, an inhibitor of metalloprotease, markedly protected against the onset of cell death during chemical hypoxia. Half-maximal protection after 60 min occurred at 0.5 microM. Phospholipase inhibitors, chlorpromazine (50 microM) and mepacrine (50 microM), also substantially retarded cell killing. U74006F, an inhibitor of lipid peroxidation, slowed cell killing to a lesser extent during chemical hypoxia and after oxidative stress with t-butyl hydroperoxide. Calciphor, a dimer of prostaglandin B1, did not protect against cell killing during chemical hypoxia or t-butyl hydroperoxide toxicity. In conclusion, this high capacity cytotoxicity assay for cells cultured in 96-well microtiter plates is suitable for rapid screening of potential cytoprotective agents in a variety of cell types.

Animals↗

PubMed1992

Fura-2 measurement of cytosolic free Ca2+ concentration in corpus allatum cells of larval Manduca sexta.

Cytosolic free Ca2+ has been implicated in the regulation of the larval corpus allatum (CA) of the tobacco hornworm Manduca sexta since agents presumed to cause changes in intracellular Ca2+ concentrations affect both basal and neuropeptide-modulated synthesis/release of the juvenile hormones. To determine whether differences in cytosolic free [Ca2+] in CA cells were associated with different levels of gland activity, methods were developed for dissociating CA cells with the retention of biosynthetic activity and for uptake by the dissociated cells of the fluorescent Ca2+ indicator Fura-2. Digitized fluorescence microscopy of the Fura-2-loaded cells enabled measurement of intracellular Ca2+ levels in individual cells. Intracellular free Ca2+ levels were measured in CA cells from selected days during the fifth larval stadium and were found to be highest when the CA were biosynthetically active. Treatment of CA cells from day 6 with the Ca2+ ionophore ionomycin elevated the intracellular Ca2+ level, corroborating the involvement of elevated intracellular [Ca2+] in ionophore effects on juvenile hormone (JH) and JH acid synthesis. The results of the study are considered in relation to our knowledge of the role of Ca2+ in the function of endocrine gland cells.

Animals↗

PubMed1992

Transforming growth factor-beta (TGF-b) induced phosphorylation of the myristoylated alanine rich C kinase substrate (MARCKS) protein in ovarian granulosa cells is modulated by follicle stimulating hormone (FSH).

The mechanism by which TGF-b1 affects granulosa cell physiology as well as the modulation of TGF-b1 activity by FSH are not understood. We tested the hypothesis that TGF-b1 exerts its effects on granulosa cells via activation of protein kinase C (PKC). Immunoprecipitation of the MARCKS protein from 32P labeled rat granulosa cells was used to assay PKC activation. 20 minute treatment with TGF-b1 (8 ng/ml), forskolin (30 microM), and TPA (200 nM) all caused an increase in MARCKS phosphorylation as quantified by densitometric scanning. FSH did not increase MARCKS phosphorylation above control levels while exposure of cells to both FSH and TGF-b1 (10 ng/ml) decreased phosphorylation of the MARCKS protein to control levels. These data suggests that (1) TGF-b1 signal transduction in rat granulosa cells may partially involve phosphorylation of the MARCKS protein; and, (2) in granulosa cells FSH can modulate TGF-b1 induced MARCKS phosphorylation.

Animals↗

PubMed1992

Quantitative precision of an automated, fluorescence-based image cytometer.

Digital image-based cytometry of clinical specimens labeled with fluorescent, disease-specific markers holds promise for becoming an important diagnostic and prognostic technique because the technique can make a diverse range of quantitative biochemical, morphologic, densitometric and contextual measurements on intact specimens. It has been previously shown by us, using an image cytometer (IC) consisting entirely of commercially available components, that the nuclei of individual cells in slide-supported specimens can be detected automatically using a fluorescent DNA stain and image analysis software. The purpose of this study was to determine the precision of the IC for quantifying the integrated fluorescence intensity and area of fluorescent standard beads and nuclei. Integrated intensities could be quantified to between 2.3% and 3.5% precision using a 40x objective lens and between 1.6% and 2.3% using a 20x objective. The main contribution to this uncertainty was 2% inaccuracy in determining the variations in sensitivity over the imaging area. Areas could be quantified to between 0.91% and 2.1% using a 40x objective and between 2.8% and 3.2% using a 20x objective. Significant quantification errors were introduced if the objects were not in focus or were touching each other. Overall, however, these results demonstrated that image cytometry of fluorescence-stained specimens can yield quantitative results with sufficient precision for determining DNA ploidy distributions and for making other measurements on clinical specimens.

3T3 Cells↗

PubMed1992

Synergism of cyclosporin A and phospholipase inhibitors in protection against lethal injury to rat hepatocytes from oxidant chemicals.

Cyclosporin A and phospholipase inhibitors block the oxidant-induced mitochondrial permeability transition. Here, protection by these agents against cytotoxicity of oxidant chemicals was evaluated in rat hepatocytes. The combination of cyclosporin A and a phospholipase inhibitor (trifluoperazine, mepacrine, or dibucaine), but neither alone, substantially protected against lethal injury from 0.5 mM iodoacetate but had little effect against iodoacetate plus 2.5 mM KCN. Against 100 microM t-butylhydroperoxide, cyclosporin A and trifluoperazine protected only if fructose was present. Cyclosporin A plus phospholipase inhibition protected slightly against 2.5 mM cystamine, but actually increased lethal injury after 100 microM menadione. These effects are consistent with an oxidant-induced mitochondrial permeability transition that accelerates cell killing after iodoacetate and t-butylhydroperoxide.

Animals↗

PubMed1991

Mechanism of cellular uptake of modified oligodeoxynucleotides containing methylphosphonate linkages.

The cellular uptake and intracellular distribution of methylphosphonate oligonucleotides (15 mers) has been examined using both 32P labeled and fluorescent labeled oligonucleotides. The cellular uptake process for methylphosphonate oligonucleotides is highly temperature dependent, with a major increase in uptake occurring between 15 and 20 degrees C. Most of the label which becomes cell associated at 37 degrees C cannot be removed by acid washing or trypsinization and thus seems to be within the cell. Visualization of rhodamine labeled methylphosphonate oligonucleotides using digital imaging fluorescence microscopy reveals a vesicular subcellular distribution suggestive of an endosomal localization. There was extensive co-localization of rhodamine labeled methylphosphonate oligonucleotides with fluorescein-dextran, an endosomal/lysosomal marker substance. The apparent endocytotic uptake of labeled methylphosphonate oligonucleotides could not be blocked by competition with unlabeled methylphosphonate or phosphodiester oligonucleotides, nor by ATP. This contrasts with the situation for radiolabeled phosphodiester oligonucleotides whose uptake can be completely blocked with unlabeled competitor. Uptake of phosphodiester oligonucleotides, but not of methylphosphonate oligonucleotides, could be blocked by acidification of the cytosol. These observations suggest that the pathway of cellular uptake of methylphosphonate oligonucleotides involves fluid phase or adsorbtive endocytosis, and is distinct from the uptake pathway for phosphodiester oligonucleotides.

Animals↗

PubMed1991

Suppression of Ca2+ oscillations in cultured rat hepatocytes by chemical hypoxia.

The model of "chemical hypoxia" with KCN plus iodoacetic acid mimics the ATP depletion and reductive stress of hypoxia. Here, we examined the effects of chemical hypoxia on cytosolic free Na+ and Ca2+ in single cultured rat hepatocytes by multiparameter digitized video microscopy and ratio imaging of sodium-binding furan indicator (SBFI) and Fura-2. Intracellular Na+ increased from about 10 mM to more than 100 mM after 20 min of chemical hypoxia, whereas cytosolic free Ca2+ remained virtually unchanged. In normoxic hepatocytes, phenylephrine (50 microM) and Arg-vasopressin (20-40 nM) induced Ca2+ oscillations in 70 and 40% of cells, respectively. These Ca2+ oscillations were suppressed after one spike following the onset of chemical hypoxia. Phenylephrine and vasopressin also increased inositol phosphate formation by 22 and 147%, respectively. This effect was suppressed by KCN plus iodoacetate. Intracellular acidosis is characteristic of chemical hypoxia. Intracellular acidosis induced by 40 mM Na-acetate suppressed Ca2+ oscillations but did not inhibit hormone-induced inositol phosphate formation. Cytosolic alkalinization also suppressed Ca2+ oscillations. However, prevention of intracellular acidosis with monensin (10 microM) did not prevent suppression of Ca2+ oscillations during chemical hypoxia. Mitochondrial depolarization with uncoupler did not change free Ca2+ levels during chemical hypoxia, indicating that mitochondria do not regulate free Ca2+ during chemical hypoxia. From these results, we conclude: 1) chemical hypoxia does not block Na+ influx across the plasma membrane; 2) Chemical hypoxia inhibits hormone-stimulated Ca2+ flux pathways across cellular membranes by two different mechanisms: (a) by ATP depletion, which disrupts hormone-myo-inositol 1,4,5-triphosphate coupling, and (b) by intracellular acidosis, which inhibits myo-inositol 1,4,5-triphosphate-stimulated Ca2+ release from intracellular stores; 3) during ATP depletion by chemical hypoxia, mitochondria do not take up Ca2+ to maintain cytosolic free Ca2+ at low concentrations.

Animals↗

PubMed1991

Protection by acidotic pH against anoxia/reoxygenation injury to rat neonatal cardiac myocytes.

We assessed the effect of acidosis on cell killing during anoxia and reoxygenation in cultured rat neonatal cardiac myocytes. After 4.5 hours of anoxia and glycolytic inhibition with 2-deoxyglucose, loss of viability was greater than 90% at pH 7.4. In contrast, at pH 6.2-7.0, viability was virtually unchanged. To model changes of pH and oxygenation during ischemia and reperfusion, myocytes were made anoxic at pH 6.2 for 4 hours, followed by reoxygenation at pH 7.4. Under these conditions, reoxygenation precipitated loss of viability to about half the cells. When pH was increased to 7.4 without reoxygenation, similar lethal injury occurred. No cell killing occurred after reoxygenation at pH 6.2. We conclude that acidosis protects against lethal anoxic injury, and that a rapid return from acidotic to physiologic pH contributes significantly to reperfusion injury to cardiac myocytes - a 'pH paradox'.

Acidosis↗

PubMed1991

Regional and mechanistic differences in platelet-derived growth factor-isoform-induced alterations in cytosolic free calcium in porcine vascular smooth muscle cells.

Three specific platelet-derived growth factor (PDGF) isoforms are thought to bind with differing affinities to two distinct PDGF receptors which undergo activation following dimerization. Recent evidence has been presented that marked differences exist between the ability of PDGF-AA versus PDGF-AB and PDGF-BB to stimulate alterations in second messengers in cultures of vascular smooth muscle cells (VSMC), a result which was thought to be due to low numbers of the A-type receptor in this cell type (Sachinidis, A., Locker, R., Vetter, W., Tatje, D., and Hoppe, J. (1990) J. Biol. Chem. 265, 10238-10243, 1990). In particular, PDGF-BB and PDGF-AB but not PDGF-AA could elicit alterations in cytosolic free calcium (Ca2+i). However, because these studies were performed on large cell populations using biochemical assays of PDGF activity, a minor PDGF-AA-Ca(2+)-responsive population of cells might go undetected. To test this possibility, VSMC were isolated from either thoracic or abdominal pig aorta, and alterations in Ca2+i were monitored using Multiparameter Digitized Video Microscopy following stimulation with PDGF isoforms alone, or either before or after exposure of VSMC to 5 mM EGTA. PDGF-AA-responsive cells were found to exist only in cultures of thoracic VSMC, caused oscillations in Ca2+i, represented 20% of the PDGF-BB-responsive cells, and were subsequently responsive to PDGF-BB. PDGF-BB elicited monophasic alterations in Ca2+i in both thoracic and abdominal VSMC. Prior addition of EGTA inhibited PDGF-AA but not PDGF-BB-induced alterations in Ca2+i. Addition of EGTA during PDGF-AA-induced Ca2+i oscillations inhibited subsequent oscillations in Ca2+i, while addition of EGTA at the peak of the PDGF-BB Ca2+ response resulted in a more rapid return of Ca2+i to prestimulation levels. These data suggest that regional differences in the distribution of PDGF-A- and B-type receptor exists in vivo, and that activation of the A- and B-type PDGF receptors results in distinct alterations in Ca2+i.

Animals↗

PubMed1991

Verapamil and cyclosporin A modulate doxorubicin toxicity by distinct mechanisms.

Cyclosporin A has been reported to enhance the sensitivity of cells displaying multiple drug resistance to anthracyclines. However, the mechanism of action of cyclosporin A in modulating drug resistance is still controversial. This study compares the effects of cyclosporin A and verapamil on doxorubicin resistance in chinese hamster ovary cells (CHRC5) using several criteria including in vitro cytotoxicity, drug accumulation, intracellular distribution by video microscopy, and nuclear DNA damage. Our results demonstrate that verapamil modulation of doxorubicin resistance was paralleled by cellular accumulation of doxorubicin, altered intracellular distribution of doxorubicin from cytoplasm to nucleus, and an increase in the formation of doxorubicin related DNA strand breaks. In contrast, the modulating effect of cyclosporin was qualitatively different. High concentrations of cyclosporin (5 micrograms/ml) increased doxorubicin accumulation and caused partial redistribution to the nucleus. However, with low concentrations of cyclosporin (1 microgram/ml) increased doxorubicin sensitivity was observed without changes in net accumulation of doxorubicin or intracellular distribution, and without enhanced doxorubicin induced DNA breakage. These results suggest that cyclosporin A can modulate doxorubicin cytotoxicity by means other than interference with the P-glycoprotein drug efflux system.

Animals↗

PubMed1991

A pH-dependent phospholipase A2 contributes to loss of plasma membrane integrity during chemical hypoxia in rat hepatocytes.

Previous studies have suggested that alterations in phospholipid composition of plasma membranes may underlie lethal cell injury due to hypoxic and ischemic injury. The present study was designed to determine if such alterations are due to the activation of a pH-dependent phospholipase A2. Loss of cell viability and phospholipase A2 activity measured by arachidonic acid release increased in parallel during metabolic inhibition with KCN and iodoacetate (chemical hypoxia). Acidosis (pH 6.5) and the phospholipase inhibitors, dibucaine and mepacrine, delayed loss of cell viability and release of arachidonic acid to a similar extent. These findings suggest that a pH-dependent phospholipase A2 causes alterations in plasma membrane phospholipid composition after ATP-depletion which contribute to lethal cell injury.

Animals↗

PubMed1991

Follicle-stimulating hormone increases c-fos mRNA levels in rat granulosa cells via a protein kinase C-dependent mechanism.

Recent evidence has been presented that follicle-stimulating hormone (FSH) stimulates the induction of granulosa cell c-fos protooncogene mRNA in vivo (Pennybacker and Herman (1989) J. Cell Biol. 109, 151A; Delidow et al. (1990) Endocrinology 126, 2302-2306), yet the mechanisms by which FSH induces c-fos mRNA expression have not been delineated. To elucidate the mechanisms of FSH-dependent c-fos mRNA expression, we measured the time and dose dependence of c-fos mRNA levels using Northern blot analysis in intact ovaries and cultured granulosa cells in response to FSH. In intact ovaries, FSH-induced c-fos mRNA expression was time dependent with maximal expression at 90 min post FSH injection, while in cultures of granulosa cells obtained from estrogen-primed immature female rats, c-fos mRNA levels were highest after 30 min exposure to FSH and at a concentration of 100 ng/ml. Neither 8-bromo adenosine 3',5'-cyclic monophosphate (8-br-cAMP), at doses ranging from 0.1 to 10 mM, nor 100 microM forskolin (in the presence or absence of 200 microM isobutyl-methylxanthine) or luteinizing hormone (LH, 100 ng/ml) were able to mimic FSH-induced c-fos mRNA expression in granulosa cell cultures. However, tetradecanoyl-13-phorbol acetate (TPA, 200 nM) was able to induce c-fos mRNA expression. The protein kinase C (PKC) inhibitors H-7 (0.3-30 microM) and staurosporine (0.75 micrograms/ml) blocked FSH-induced c-fos mRNA expression in cultured granulosa cells while HA 1004, an inhibitor of cGMP- and cAMP-dependent protein kinases at 30 microM had no effect on TPA-induced c-fos expression, and only minimally inhibited FSH-induced c-fos expression. Both FSH (100 ng/ml) and forskolin (3 microM) increased progesterone production in cultured granulosa cells. These data support the hypothesis that FSH specifically induces c-fos mRNA expression by a PKC-dependent mechanism and that the cAMP arm of the FSH response pathway is operant in these cells.

Animals↗

PubMed1991

Lipid order in hepatocyte plasma membrane blebs during ATP depletion measured by digitized video fluorescence polarization microscopy.

Low-light digitized video fluorescence polarization microscopy was used to measure lipid order parameters in plasma membrane blebs of single, cultured rat hepatocytes during ATP depletion with the metabolic inhibitors cyanide and iodoacetic acid. Hepatocytes were labeled on the microscope stage with the plasma membrane probe trimethylammoniumdiphenylhexatriene at successive stages of cell injury. A pair of fluorescence polarization ratio images was obtained from a series of four fluorescence images recorded with a polarizer in the emission path oriented first parallel and then perpendicular to each of two orthogonal excitation light polarization directions. From the polarization ratio images, the lipid order parameter S was determined in individual plasma membrane blebs. Results indicate that the plasma membrane becomes uniformly rigid within a few minutes of the addition of metabolic inhibitors when small surface blebs have formed and ATP levels have fallen by greater than 95%. The measured order parameter of S approximately 0.95 in plasma membrane blebs, compared with S approximately 0.75 in normoxic cell plasma membranes, remained unchanged throughout the course of bleb development and ultimate cell death. These findings demonstrate that significant alteration in hepatocyte plasma membrane structure occurs early in hypoxic cell injury.

Adenosine Triphosphate↗

PubMed1991

Cytosolic free Ca2+ and proteolysis in lethal oxidative injury in endothelial cells.

Oxygen free radicals (OFR) are thought to mediate ischemia-reperfusion injury to endothelium of heart, lung, brain, liver, and kidney and contribute to development of atherosclerosis, pulmonary O2 toxicity, and adult respiratory distress syndrome. Increased cytosolic free Ca2+ (Cai2+) has been proposed as a mechanism of injury from oxidative stress, yet the pathways by which an increase in Cai2+ may cause OFR-mediated endothelial cell injury remain unknown. Using multiparameter digitized video microscopy and the fluorescent probes, fura-2 acetoxymethyl ester and propidium iodide, we measured Cai2+ and cell viability in human umbilical endothelial cells during oxidative stress with xanthine (50 microM) plus xanthine oxidase (40 mU/ml). Oxidative stress caused a sustained increase in Cai2+ from a resting level of 90-100 nM to near 500 nM, which was preceded by formation of plasma membrane blebs. The increase in Cai2+ was prevented by removal of extracellular Ca2+ (Cao2+). Prevention of the increase in Cai2+ was associated with prolonged cell viability. Readdition of Cao2+ resulted in an immediate large increase in Cai2+ and rapid onset of cell death. The protease inhibitors, leupeptin and pepstatin, delayed the increase in Cai2+ and prolonged cell viability. The results are consistent with the hypothesis that endothelial cell injury due to oxidative stress may be the result of Cai2+ influx and resultant activation of Ca(2+)-dependent proteases.

Calcium↗

PubMed1991

Automated image-based cytometry with fluorescence-stained specimens.

The combination of digitized microscopy, algorithms for object recognition and fluorescent labeling is a promising approach for reliable, quick, automated and cost-effective screening of clinical specimens. We describe two conceptually different algorithms for detecting objects in fluorescence microscopic images. One, which is partially automated, compares a mask that represents a typical object with every position in the image; the other, which is fully automated, calculates threshold intensities to segment the image into regions of objects and background. Applications of the algorithms in conjunction with a prototype image-based cytometer are demonstrated for determining the DNA ploidy distribution of cultured human endometrial cells and determining the DNA ploidy distribution and the fraction of cells expressing the E6 antigen of human papilloma virus serotypes 16 and 18 in a PAP smear. The encouraging results from this study suggest that automated image-based cytometry utilizing fluorescent stains will be a valuable asset for clinical screening.

Algorithms↗

PubMed1991

Automated fluorescence image cytometry. DNA quantification and detection of chlamydial infections.

Digitized fluorescence microscopy in conjunction with automated image segmentation is a promising approach for screening clinical specimens quickly and reliably. This paper describes the hardware and software of a prototype image-based cytometer that can identify fluorescent objects, discriminate true objects from artifacts and divide overlapping pairs of objects. The use of this image cytometer is discussed for: (1) the measurement of the DNA ploidy distribution of isolated mature rat liver nuclei labeled with 4',6-diamidine-2-phenylindole; (2) the comparison of the DNA ploidy distributions of the same samples measured by image cytometry (ICM) and flow cytometry (FCM); and (3) the quantification of chlamydial infection by double labeling cells with antichlamydiae antibody and Hoechst 33258 for nuclear DNA analysis. Ploidy distributions measured by the automated image cytometer compared favorably to those obtained by FCM. All pairs of overlapping nuclei were automatically detected by an additional computer algorithm, and those pairs that were clearly more than one nucleus by visual inspection were correctly divided. The irregular morphology of the chlamydiae-infected cells meant that 26% of them were not correctly identified in the fluorescein-stained images (as judged by manual inspection), but all cells were nevertheless detected correctly from the images of the Hoechst-stained samples. Automated fluorescence ICM yielded results similar to those obtained with FCM and had the additional benefit of maintaining cell and tissue architecture while preserving the opportunity for subsequent manual inspection of the specimen.

Algorithms↗

PubMed1991

Recovery of cultured rat neonatal myocytes from hypercontracture after chemical hypoxia.

Cardiac myocytes exposed to anoxia hypercontract into a blebbed, rounded mass. Such hypercontraction is usually considered a manifestation of irreversible injury. Here, we studied functional recovery, long-term viability and ATP levels of cultured neonatal rat cardiac myocytes after metabolic inhibition with cyanide and 2-deoxyglucose, a model of 'chemical hypoxia' which mimics the ATP depletion and reductive stress of hypoxia. After addition of inhibitors, 5-day cultured myocytes ceased spontaneous contractions within 1-2 min, blebbed and hypercontracted after 35 min, and lost viability after 100 min as assessed by nuclear labelling with propidium iodide. 11-day cultured myocytes exhibited a similar progression of injury. When the metabolic inhibitors were removed, spontaneous contractions resumed after an average of 11 min in non-hypercontracted myocytes. In hypercontracted myocytes, weak contractions resumed after 35 min, regardless of the length of time spent in the hypercontracted state, but restoration of strong synchronous contractions took hours. Recovering cells remained viable and exhibited spontaneous contractions through 24 hours of observation, whereas contractility never returned in propidium iodide-labelled cells. ATP levels decreased rapidly after chemical hypoxia and were partially restored upon washout of inhibitors. ATP recovery was similar in non-hypercontracted and hypercontracted cells. Thus, contractile dysfunction during recovery in hypercontracted cells was not due to lack of regeneration of ATP. In conclusion, hypercontracture is not a manifestation of irreversible injury. Contractile dysfunction following recovery from hypercontracture observed here may represent 'in vitro stunning'.

Adenosine Triphosphate↗