Digital Imaging Fluorescence Microscopy Research Articles

Membrane domains containing phosphatidylserine and substrate can be important for the activation of protein kinase C

The relationship between lipid domains and enzyme activity was studied via the direct visualization and quantitation of domains by fluorescence digital imaging microscopy. The substrate used in these experiments was a basic peptide derived from a prominent cellular substrate (MARCKS) of protein kinase C. The MARCKS peptide and phosphatidylserine, which were labeled by two different fluorophores, colocalized into domains in large vesicles (5-10 microns). Increasing the ionic strength disrupted the domains of the MARCKS peptide and phosphatidylserine, and this was accompanied by a decrease in protein kinase C activity. Dansylpolylysine, which inhibits protein kinase C, was similar to the MARCKS peptide in forming domains enriched in phosphatidylserine. The degree of enrichment of the MARCKS peptide in the phosphatidylserine domains decreased proportionally with protein kinase C activity when polylysine was added. Polylysine caused the MARCKS peptide to be displaced from the domains into the nondomain areas of the vesicles. This suggested that binding of the substrate to the vesicles was not the critical factor for protein kinase C activity, but rather it was the organization of the substrate into domains that was related to the activation of the enzyme. Gramicidin, which was chosen to represent a neutral membrane protein, was excluded from the domains with phosphatidylserine, and it had no effect on the enrichment of the domains or the enzyme activity. The results of this study show that the formation of membrane domains can be important for the activation of protein kinase C and the activity can be inhibited by disrupting the domains.

Cell cycle analysis of amount and distribution of nuclear DNA topoisomerase I as determined by fluorescence digital imaging microscopy.

Fluorescence digital imaging microscopy (FDIM) has been used to perform a cell cycle analysis of both the amount and the distribution of nuclear DNA topoisomerase I in individual CEM human leukemia cells. Cells were stained by indirect immunofluorescence methods using a polyclonal antiserum generated with a 21-amino-acid peptide representing amino acids 219-239 of human topoisomerase I. Immunohistochemical staining was followed by staining with Hoechst dye 33342, allowing DNA content to be determined in each cell. Cell cycle analysis showed that nuclear topoisomerase I content doubled (2.2-fold increase) as the cells progressed from G1 to G2/M phases of the cell cycle. However, when normalized for nuclear size, topoisomerase I content per nuclear area remained almost constant (1.3-fold increase). For comparison, we measured the amount of proliferating cell nuclear antigen (PCNA), a protein whose expression fluctuates during the cell cycle. Nuclear PCNA content increased 2.7-fold from G1 to S phase, then declined in G2/M- phases, whereas PCNA content per nuclear area increased 1.7-fold from G1 to S phase. We also measured topoisomerase I content in leucine-deprived cells to determine if altered growth conditions affect topoisomerase I protein expression. Compared to CEM cells in logarithmic growth, leucine-deprived CEM cells had 1.8-fold less topoisomerase I content per nuclear area. Subnuclear distribution studies of proliferating CEM cells showed topoisomerase I to be localized predominantly in the nucleoli throughout the cell cycle. In contrast, leucine-deprived cells exhibited a perinuclear distribution of topoisomerase I. Our results show that FDIM is a useful technique in determining the cell cycle position and both the content and the distribution of topoisomerase I as well as other nuclear proteins in individual cells.

Depletion and filling of intracellular calcium stores in vascular smooth muscle

In vascular smooth muscle, binding of vasoactive substances to surface membrane receptors leads to a rise of intracellular cytoplasmic Ca2+ and to contraction. Cytoplasmic free Ca2+ concentration ([Ca2+]i) increases through release of Ca2+ from intracellular stores and Ca2+ entry through surface membrane ion channels. Membrane-permeant and membrane-impermeant forms of fura 2 were used to distinguish changes in intracellularly stored Ca2+ ([Ca2+]s) from changes in [Ca2+]i. The spatiotemporal patterns of the movement of Ca2+ between these two cellular compartments in cultured vascular smooth muscle cells (A7r5 cell line) were visualized with digital imaging fluorescence microscopy. The Ca2+ stores were localized by double staining with a fluorescent organelle-specific dye and the Ca2+ indicator. [Ca2+]s was measured after accumulation of the membrane-permeant form of fura 2 inside the stores and quenching of the fura 2 fluorescence in the cytoplasmic compartment with manganese. Stimulation with vasopressin led to a transient increase of [Ca2+]i and a concomitant decrease of [Ca2+]s. After stimulation with vasopressin, [Ca2+]i returned rapidly to normal resting levels, whereas the recovery of [Ca2+]s occurred on a much slower time scale. The refilling pathway of depleted stores involved Ca2+ entry into the bulk cytoplasmic compartment before uptake into the stores.

Differential cytotoxicity in mouse epidermal JB6 cells: A potential mechanism for oxidant tumor promotion

It has been suggested that superior antioxidant defense systems protect promotion-sensitive (p+t) mouse epidermal JB6 clone 41 cells from excessive deleterious effects of oxidants, allowing their clonal expansion in contrast to that of promotion-resistant (p-) clone 30 cells. In support of this concept, we report that oxidants produced by xanthine/xanthine oxidase cause more cytotoxicity, cellular damage, and cell death in p-cells. Cell surface blebbing, an early morphological consequence of oxidative injury, was detected in cultures grown on glass coverslips. While a rise in cytosolic ionized calcium ([Ca2+]i) preceding bleb formation was observed in both p+ and p- cells by digital imaging fluorescence microscopy, elevated levels of [Ca2+]i were sustained longer in p- cells. This increase was dependent on the levels of extracellular ionized calcium ([Ca2+]e) in p+ but not p- cells. We conclude that the superior antioxidant defense or improved Ca2+ buffering of promotable clone 41 cells protects them from more severe deregulation of [Ca2+]i and, as a consequence, from excessive cytotoxicity after exposure to oxidant promoters.

Cytosolic-free calcium elevation in Trypanosoma cruzi is required for cell invasion.

To replicate, the trypomastigote form of Trypanosoma cruzi must invade host cells. Since a role for Ca2+ in the process of cell invasion by several intracellular parasites has been postulated, changes in the intracellular Ca2+ concentration in T. cruzi trypomastigotes and in tissue culture L6E9 myoblasts during their interaction were studied at the single cell level using digital imaging fluorescence microscopy or in cell suspensions by fluorescence spectrophotometry. An increase in cytosolic Ca2+ in T. cruzi trypomastigotes was detected at the single cell level after association of the parasites with the myoblasts. Ca2+ mobilization in the host cells was also detected upon contact with trypomastigotes either at the single cell level or in cells grown in coverslips and exposed to suspensions of trypomastigotes. Pretreatment of the parasites with the Ca2+ chelators quin 2 (50 microM) or bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA, 50 microM) decreased the trypomastigotes' association to myoblasts by approximately 40 and 63%, respectively, thus indicating that an increase in intracellular Ca2+ concentration in the parasites is required for cell invasion in addition to Ca2+ mobilization in the host cells.

Characterization of signaling pathways to Na+/H+ exchanger activation with epidermal growth factor in hepatocytes.

To investigate the signaling pathways to Na+/H+ exchanger activation with epidermal growth factor in hepatocytes, we measured changes in cytosolic free calcium and intracellular pH levels at the single-cell level using digital imaging fluorescence microscopy of fura-2- or BCECF-loaded hepatocytes in primary culture. Epidermal growth factor induced cytosolic free calcium oscillations consisting of periodic trains of spikes with a latency period of up to several minutes. These calcium responses were inhibited by tyrosine kinase inhibitor genistein (100 mumol/L) and abolished by emptying of intracellular Ca2+ pools with 3 mumol/L thapsigargin, an inhibitor of Ca(2+)-ATPase on the endoplasmic reticulum. Epidermal growth factor (1 nmol/L) induced an intracellular pH increase of 0.12 +/- 0.07 units from the basal level of 7.25 +/- 0.09 units after several minutes of latency. This effect was completely abolished by 1 mmol/L amiloride, an inhibitor of the Na+/H+ exchanger. The epidermal growth factor-induced intracellular pH increase was inhibited by pretreatment of hepatocytes with genistein (100 mumol/L), thapsigargin (3 mumol/L) or calmodulin inhibitor W-7 (25 mumol/L), but not with protein kinase C inhibitor H-7 (50 mumol/L) or with cyclic AMP-dependent kinase inhibitor H-8 (60 mumol/L). Phorbol ester PMA (phorbol 12-myristate 13-acetate), a potent activator of protein kinase C, induced a slight intracellular pH increase significantly smaller than that with epidermal growth factor, whereas this effect was completely blocked by pretreatment with H-7, indicating that PMA-induced intracellular pH increase is mediated by protein kinase C pathways, unlike epidermal growth factor.(ABSTRACT TRUNCATED AT 250 WORDS)

TUMOR VASCULAR SHUTDOWN FOLLOWING PHOTODYNAMIC THERAPY BASED ON POLYHEMATOPORPHYRIN OR 5-AMINOLEVULINIC ACID

The effects of photodynamic therapy (PDT) based on polyhaematoporphyrin (PHP) or 5-aminolaevulinic acid (ALA) on the tumour vascular perfusion (TVP) of a rat fibrosarcoma were examined using an (RbCl)-Rb-86 extraction technique. Both forms of therapy induced large and highly significant reductions in TVP, countering the previous suggestion that ALA-PDT has no vascular effects. Ultrasensitive digital imaging fluorescence microscopy indicated that ALA-induced PpIX was distributed relatively evenly throughout the tumour. Use of Hoechst 33342 as a vascular marker revealed that PpIX was also present in cells lining the tumour microvascular spaces, contrary to previous findings.

Colocalization of Nestin and Vimentin/Desmin in Skeletal Muscle Cells Demonstrated by Three-Dimensional Fluorescence Digital Imaging Microscopy

During skeletal muscle development three intermediate filament proteins are expressed: nestin, vimentin, and desmin. Vimentin and desmin belong to the class III intermediate filaments and are closely related to each other, whereas nestin is a more distantly related, class VI, intermediate filament. It was previously observed by conventional immunocytochemistry that the intracellular patterns of nestin, desmin, and vimentin appeared indistinguishable, despite nestin's more distant evolutionary relationship. We here extend this analysis by applying three-dimensional fluorescence digital imaging microscopy to compare the intracellular distribution of nestin with that of desmin, vimentin, actin, and tubulin in G6 human fetal skeletal muscle cells. We show that in vitro differentiation of G6 cells can produce an intermediate filament expression pattern similar to that observed during myogenesis in vivo, i.e., downregulation of vimentin but not of nestin and desmin during myotube maturation. The image analysis demonstrated that the degree of overlap between nestin and desmin/vimentin was very extensive in myoblasts and in multinucleate myotubes in all regions of the cells. In contrast, nestin did not colocalize with tubulin or actin in G6 myoblasts. In particular, nestin immunoreactivity was not detected at the microtubule-organizing center, and it was only sparsely observed at the cell periphery where actin stress fibers were seen. Our data lend further support to the notion that nestin interacts very closely with the two more distantly related class III intermediate filament proteins desmin and vimentin in the entire muscle cell, before and after myotube formation. A comparison of conserved amino acid residues in the different IFs suggests that charged amino acid residues in the α-helical rod domain may play a role in the interaction.

Regulation of intracellular pH in crypt cells from rabbit distal colon

H+ secretory mechanisms and intrinsic intracellular buffering capacity were studied in crypt cells from rabbit distal colon. To this end crypts of Lieberkühn were isolated by microdissection, and intracellular pH (pHi) was measured using digital imaging fluorescence microscopy and the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)- 5(6)-carboxyfluorescein. In the absence of HCO(3-)-CO2 and presence of Na+, resting pHi was 7.51 +/- 0.04 (n = 237/23, cells/crypts). However, 6 min after superfusion with a solution containing zero Na+, 1 x 10(5) M Sch-28080 and 5 x 10(-8) M bafilomycin A1, pHi in cells at the bottom of the crypts was significantly reduced, whereas pHi in cells at the top of the crypts remained unchanged. The intrinsic buffering capacity of cells from the middle to the top portion of crypts was significantly higher in the pHi range 7.2-7.6 than of cells at the bottom of the crypt. H+ secretion after an NH(4+)-NH3 pulse amounted to 245 +/- 53 microM/s (n = 73/7) at pHi 7.1 and was largely Na+ dependent and ethylisopropylamiloride sensitive. The Na(+)-independent recovery of pHi after an acid load was insensitive to Sch-28080 and bafilomycin A1. In conclusion, pHi in colonic crypt cells is regulated through Na+/H+ exchange activity in the absence of HCO3-. In addition, intracellular buffering capacity varied with the position along the crypt axis, whereas Na+/H+ exchange activity and pHi did not.

Visualization and quantification of glycolipid polarity dynamics in the plasma membrane of the mammalian spermatozoon

Seminolipid (sulphogalactosylalkylacylglycerol), the glycolipid that is specific for mammalian germ cells, is located exclusively in the outer leaflet of the sperm plasma membrane. In this study the lateral distribution of seminolipid on sperm heads has been investigated by indirect immunofluorescence labelling and detection with digital imaging fluorescence microscopy. In freshly ejaculated sperm cells this glycolipid was present primarily at the apical ridge subdomain of the plasma membrane of the sperm head. After binding the sperm cells to zona-coated coverslips seminolipid migrated, in 40 minutes, from the apical ridge to the equatorial subdomain of the plasma membrane. A similar redistribution of seminolipid was observed during capacitation of sperm cells in vitro induced by Ca2+ or bovine serum albumin. Comparable migration of seminolipid was also found after prolonged storage of ejaculated sperm cells, albeit at a much slower rate. Addition of arylsulphatase A, an enzyme present in seminal plasma that desulphates seminolipid, significantly enhanced the migration of seminolipid during storage of sperm cells. Its breakdown product desulphoseminolipid (galactosylalkylacylglycerol) appeared highly specifically at the equatorial segment. The measured fluorescence intensity over the sperm head surface correlated linearly with the spatial probe distribution as was checked by fluorescence lifetime imaging microscopy. This paper demonstrates and quantifies for the first time the polarity of seminolipid on the surface of the sperm cell and the dynamic alterations that occur in this polarity during post-ejaculatory events.

Oxidative stress-induced perturbations of calcium homeostasis and cell death in cultured myocytes: role of extracellular calcium.

The role of extracellular calcium in the process of oxidative stress-induced calcium overload and cell death was investigated in cultured neonatal rat myocytes. Oxidative stress was induced by addition of cumene hydroperoxide (CHPO), a toxic organic hydroperoxide, in combination with varying extracellular calcium concentrations (1. normal calcium buffer: 2.5 mM Ca2+, 2. low calcium buffer: 5 microM Ca2+, 3. zero calcium buffer: 2.5 mM EGTA, no CaC12). Intracellular free calcium ion concentration ([Ca2+]i) was measured with fura-2 using a spectrofluorometer. To study the toxicant-induced changes in [Ca2+]i in more detail, single cell imaging was performed using digital imaging fluorescence microscopy (DIFM). In control experiments (in the absence of CHPO, but under different extracellular Ca2+ conditions) the [Ca2+]i remained at the basal level and cell viability was preserved. Administration of CHPO (50 microM) to the myocyte cultures generated a sustained increase in [Ca2+]i followed by loss of cell viability. A low extracellular calcium concentration (5 microM), in the absence or presence of diltiazem (10 microM), induced a delay in the rise in [Ca2+]i but was not able to prevent the CHPO-induced calcium overload and cell necrosis. Addition of EGTA (2.5 mM) to the low calcium buffer resulted also in CHPO-induced cell death, although no increase in [Ca2+]i was observed. In normal and low calcium buffers, DIFM revealed that CHPO produced a temporally and spatially heterogeneous distribution of [Ca2+]i in a group of myocytes. So, in the presence of normal or low extracellular Ca2+, CHPO intoxication of cultured myocytes leads to an increase of [Ca2+]i prior to the onset of cell death. If extracellular Ca2+ is chelated by EGTA, CHPO also induces cell death which is not preceded by cellular calcium overload. Apparently a disturbance in the calcium homeostasis is not causally related with oxidative stress-induced myocardial cell death.

Effects of Ca2+ channel blockers, low Ca2+ medium and glycine on cell Ca2+ and injury in anoxic rabbit proximal tubules

L-type Ca2+ channel blockers (CCBs) have been shown to be protective against ischemia-induced injury of the kidney, suggesting that increased intracellular Ca2+ levels ([Ca2+]i) play an important role in the pathogenesis of ischemic cell injury. To assess the role of [Ca2+]i in anoxic injury of the proximal tubule (PT) and the protective effect of CCBs, digital imaging fluorescence microscopy was used to monitor [Ca2+]i in individual PT cells during 60 minutes of anoxia. [Ca2+]i started to rise within 10 minutes and reached maximal levels between 30 to 45 minutes of anoxia. The onset of this increase and the maximal levels reached varied markedly among individual cells. The mean values for initial and maximal anoxic [Ca2+]i were 109 +/- 2 (N = 209) and 422 +/- 14 (N = 240) nM, respectively. Methoxyverapamil (D600; 1 microM) significantly reduced anoxic [Ca2+]i to 122 +/- 5 nM (P < 0.05; N = 79). Removal of extracellular Ca2+ completely abolished anoxia-induced increases in [Ca2+]i, confirming that these increases in [Ca2+]i result from Ca2+ influx. During 60 minutes of anoxia, PT cells showed a gradual decrease in cell viability to 54 +/- 2%. D600 (1 microM) significantly increased cell viability to 64 +/- 3% (P < 0.05). Glycine (5 mM), however, increased cell viability to 77 +/- 4% without a significant reduction in anoxic [Ca2+]i levels. Low Ca2+ medium only protected when 0.1 mM La3+ was included, a condition which increased cell viability to 82 +/- 5%.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Benzene Metabolites on Receptor-Mediated Phagocytosis and Cytoskeletal Integrity in Mouse Peritoneal Macrophages

Exposure to benzene can induce a number of hematotoxicities and decrease host resistance to microorganisms and tumors. Several studies have shown that metabolism of benzene to reactive intermediates is required for myelotoxicity. Since receptor-mediated phagocytosis by macrophages is an important host defense, we have examined the effects of benzene metabolites on receptor-mediated phagocytosis in cultured murine peritoneal macrophages. 1,4-Benzoquinone (BQ) was the most potent of the metabolites examined. Ten-minute exposures to a 12.5 μM concentration inhibited Fc and complement receptor-mediated phagocytosis by ≥90%. Macrophage viability was largely unaffected by BQ treatment. Exposure to 50 and 100 μM 1,2,4-benzenetriol (BT) inhibited Fc receptor-mediated phagocytosis by 70 and 95%, respectively. Hydroquinone (HQ) elicited a major decrease (50%) only at 100 μM. The comparative inhibitory potencies of BT and HQ correlate with previously published data on their relative facility for autooxidation to quinones at physiological pH. Catechol had no effect at the concentrations employed. Macrophages treated with BQ and BT failed to recover their Fc receptor-mediated phagocytic capacity when incubated overnight in the absence of the xenobiotics. Only small differences in the inhibition of Fc receptor-mediated phagocytosis were observed between macrophages exposed to BQ at 4 versus 37°C. BQ also had little effect on the Fc receptor binding of target cells. Fluorescent digital imaging microscopy demonstrated that BQ treatment markedly decreased the filamentous actin content of macrophages. However, BQ bound in low amounts to purified actin and did not affect its assembly. Our findings suggest that a mechanism for inhibition of Fc receptor-mediated phagocytosis by BQ is disruption of filamentous actin via an effect(s) other than the direct alkylation of actin by BQ.

Endogenous ganglioside GM1 modulates L-type calcium channel activity in N18 neuroblastoma cells

Digital imaging fluorescence microscopy was used to investigate the effect of the B subunit of cholera toxin on calcium homeostasis in neuroblastoma N18 cells. The B subunit, which binds specifically to ganglioside GM1 in the outer leaflet of the cell membrane, was found to induce a sustained increase of intracellular calcium concentration ([Ca2+]i). The increase in [Ca2+]i was not observed in the absence of extracellular calcium, or in the presence of the calcium chelator EGTA, and was blocked by nickel. The B subunit was also found to induce an influx of manganese ions, as indicated by a quench of the intracellular fura-2 fluorescence. These data suggest that the B subunit induces an increase in calcium influx in N18 cells. Potassium-induced depolarization also stimulated manganese influx; however, after the onset of depolarization-induced influx, the B subunit had no further effect. This occlusion suggests involvement of voltage-dependent calcium channels. Treatment with BayK8644, a dihydropyridine agonist selective for L-type calcium channels, induced manganese influx that was not altered by the B subunit and apparently blocked the effect of the B subunit itself. Furthermore, the dihydropyridine L-type channel antagonists niguldipine or nicardipine completely inhibited B subunit-induced manganese influx. Thus, the B subunit-induced manganese influx is likely due to activation of an L-type voltage-dependent calcium channel. Spontaneous influx of manganese ions was also inhibited by nicardipine or niguldipine and by exogenous gangliosides. Ganglioside GM1 was more potent than GM3, but globoside had no significant effect. The modulation of L-type calcium channels by endogenous ganglioside GM1 has important implications for its role in neural development, differentiation, and regeneration and also for its potential function in the electrical excitability of neurons.

Role of cytosolic Ca2+ and protein kinases in the induction of the hsp70 gene

The role of cytosolic Ca2+ ([Ca2+]i) and protein kinases in the hsp70 induction following heat shock was investigated in cultured rat proximal tubular epithelial (PTE) cells. Changes in [Ca2+]i were measured by digital imaging fluorescence microscopy using fura 2. Steady state levels of hsp70 mRNA were examined by either Northern or dot blot analyses. [Ca2+]i increased within 10 minutes and continued to increase following heat shock. The increases in [Ca2+]i were reduced in nominally Ca(2+)-free media with or without EGTA. [Ca2+]i also increased within 0.5 minutes following ionomycin, but then declined to normal levels by 1.0 to 1.5 minutes. Heat shock induced hsp70 mRNA within 15 minutes, which continued to increase up to three hours. Ionomycin also induced hsp70 mRNA, which peaked at 30 minutes, and gradually decreased thereafter. The hsp70 induction following heat shock was attenuated when extracellular Ca2+ was reduced. Chelation of [Ca2+]i by quin-2 also reduced the hsp70 induction. Inhibitors of protein kinases, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), calphostin C, genistein, and 2-aminopurine, also had inhibitory effects on the hsp70 induction. In contrast, a calmodulin inhibitor, chlorpromazine, had little effect. These results suggest that heat shock increases [Ca2+]i in rat PTE cells and that [Ca2+]i and protein kinases are involved in the hsp70 induction following heat shock.

Effects of Ca 2+ deregulation on mitochondrial membrane potential and cell viability in nucleated cells following lytic complement attack

We have previously shown [Papadimitriou JC. Ramm LE. Drachenberg CB. Trump BF. Shin ML. (1991) J. Immunol., 147, 212–217] that formation of lytic C5b-9 channels on Ehrlich ascites tumor cells induced rapid depletion of adenine nucleotides associated with prelytic leakage preceding cell death. Extracellular Ca 2+ concentration ([Ca 2+] e) reduction by chelation markedly delayed the onset of cell death, although the adenine nucleotide leakage was enhanced. In the present study, we examined the temporal relationships between ionized cytosolic Ca 2+ ([Ca 2+] i), mitochondrial membrane potential (Δψ m) and cell death in individual cells by digital imaging fluorescence microscopy (DIFM), during the earliest phase of C5b-9 attack. The results showed an immediate, > 20-fold rise in [Ca 2+] i, rapidly followed by dissipation of Δψ m and subsequent acute cell death. These events were markedly delayed by chelation of Ca 2+ e, but not by nominally Ca 2+ free medium. Differing from previous reports indicating propidium iodide labeling of viable cells bearing C5b-9 channels, with DIFM we observed nuclear fluorescence with that marker only in association with cell death. These findings indicate that Ca 2+ influx through lytic C5b-9 channels is responsible for the massive increase in [Ca 2+] i, as well as for the rapid loss of Δψ m, followed by acute cell death. When this [Ca 2+] i increase is prevented, the cell death is probably related to metabolic depletion.

Age-related variations in transport properties of the rabbit arterial wall near branches

Lipid accumulation in the human aorta occurs predominantly downstream of branches in foetuses, neonates and infants but upstream at later ages. The lipid in these deposits may derive from plasma lipoproteins. We have examined uptake of plasma proteins by the rabbit aortic wall near branches as a function of age. Albumin was labelled with a fluorescent dye and introduced into the circulation of animals fed a normal diet. The aorta was fixed in situ 3 h later and the distribution of tracer in sections through the wall was measured by using digital imaging fluorescence microscopy. Net uptake by the intima-media was higher downstream of intercostal ostia than upstream in young animals but this difference decreased and then reversed with age. Furthermore, the average of uptake by both regions was higher shortly after weaning than at later ages. These age-related variations in transport properties may explain discrepancies between previous studies of uptake, resolve apparent inconsistencies between the properties of rabbit and human arteries and, if applicable to man, might account for the non-uniform and changing pattern of lipid accumulation around arterial branches.

Measurement of absolute tracer concentrations in tissue sections by using digital imaging fluorescence microscopy. Application to the study of plasma protein uptake by the arterial wall

Digital imaging fluorescence microscopy (DIFM) of tissue sections was used to quantify uptake of labelled plasma proteins by the arterial wall. Several aspects of the measuring system were investigated so that absolute tracer concentrations and their local variation could be derived from digitized images. These investigations may be relevant to other studies employing DIFM. Nonlinearities were found to arise from offsets in the video digitizers, from background fluorescence and stray light within the microscope and from the transfer characteristics of the intensified CCD camera. Camera gain controls showed complex behaviour. Camera output fell substantially for several hours after switching on and was affected by room temperature. Large spatial variations in response were caused by the geometry of the microscope optics and by the image intensifier. However, the ratios between areas were not affected by light intensity or camera gain settings. Measured intensities were independent of the depth-wise location of fluorophores within tissue sections but they were affected by the emission from objects outside the measuring area. Photobleaching of tracer varied significantly over the range of excitation intensities and durations used but was not concentration dependent. Methods used to correct these effects and obtain a uniform, linear and constant relationship between concentration and grey level are described. Using the system and appropriate corrections, in vivo uptake of sulphorhodamine-B-labelled serum albumin by the rabbit aortic wall was investigated. Results obtained for the mean uptake of tracer and its local variation were quantitatively similar to those previously obtained with nonmicroscopic methods.

Distributions of proteins and lipids in the erythrocyte membrane.

Fluorescence digital imaging microscopy was used to characterize erythrocyte membrane domains. To investigate the role of specific proteins in forming lipid domains, erythrocyte ghosts were double-labeled with fluorescent phospholipids and a fluorescent label specific for band 3, the major integral membrane protein. Areas of enrichment, or domains, were observed for both the protein and lipid components. The size and enrichment of the protein and lipid domains in the membrane depended upon the ionic strength but not the temperature. Also, there was a higher correlation between the areas of band 3 and phosphatidylcholine enrichment than between band 3 and phosphatidylserine enrichment. This is consistent with band 3 playing a role in the formation of the phosphatidylcholine domains. Furthermore, areas of spectrin enrichment also were observed in immunofluorescently labeled ghosts, and the band 3 domains were found to coincide with the spectrin domains. Thus, the distribution of band 3 in ghosts is influenced by interactions with the cytoskeleton. Together, these experiments illustrate the type of membrane heterogeneity present in erythrocyte ghosts and the type of lipid and protein interactions that can affect the domains.

Pyrularia thionin binding to and the role of tryptophan-8 in the enhancement of phosphatidylserine domains in erythrocyte membranes.

Pyrularia thionin is a small, strongly basic peptide which interacts readily with cellular and synthetic membranes. With cells it induces hemolysis, depolarizes the cellular membrane with an accompanying influx of Ca2+, and activates an endogenous phospholipase A2. Evidence points toward a binding site involving phosphatidylserine (PS). This study shows that addition of the peptide to erythrocyte membranes as well as to vesicles formed from phospholipids isolated from erythrocyte membranes causes an enhancement of phospholipid domains which are made visible by the use of fluorescence digital imaging microscopy with fluorescent derivatives of PS (NBD-PS) and phosphatidylcholine (NBD-PC). Addition of thionin caused a large increase in NBD-PS domains, with an accompanying enrichment of NBD-PC in another separate domain. Double-labeling experiments performed with a Texas Red derivative of thionin show that the peptide binds to the domain enriched in NBD-PS. P thionin inactivated by modification of Trp-8 with N-bromosuccinimide lost the ability to enhance PS domains, although it bound to the membrane with the same affinity as native P thionin. This shows that binding to the membrane is not in itself sufficient to cause the NBD-PS and NBD-PC redistribution into domains.