Subcellular characterization of glucose uptake in coronary endothelial cells.

Despite all the evidence linking glucose toxicity to an increased risk of cardiovascular diseases, very little is known about the regulation of glucose uptake in endothelial cells. We have previously reported an asymmetric distribution of the GLUTs (1-5) and SGLT-1 in en face preparations of rat coronary artery endothelia [Gaudreault N., Scriven D.R., Moore E.D., 2004. Characterisation of glucose transporters in the intact coronary artery endothelium in rats: GLUT-2 upregulated by long-term hyperglycaemia. Diabetologia 47(12),2081-2092]. We assessed this time, through immunocytochemistry and wide field fluorescence microscopy coupled to deconvolution, the presence and subcellular distribution of glucose transporters in cultures of human coronary artery endothelial cells (HCAECs). HCAECs express GLUT-1 to 5 and SGLT-1, but their subcellular distribution lacks the luminal/abluminal asymmetry and the proximity to cell-to-cell junctions observed in intact endothelium. To determine the impact of the transporters' distribution on intracellular glucose accumulation, a fluorescent glucose analog (2-NBDG) was used in conjunction with confocal microscopy to monitor uptake in individual cells; the arteries were mounted in an arteriograph chamber with physiological flow rates. The uptake in both preparations was inhibited by cytochalasin-B and d-glucose and stimulated by insulin, but the distribution of the incorporated 2-NBDG mirrored that of the transporters. In HCAEC it was distributed throughout the cell and in the intact arterial endothelium it was restricted to the narrow cytosolic volume adjacent to the cell-to-cell junctions. We suggest that the latter subcellular organization and compartmentalization may facilitate transendothelial transport of glucose in intact coronary artery.

Asymmetric subcellular distribution of glucose transporters in the endothelium of small contractile arteries.

The authors have recently reported the presence and asymmetric distribution of the glucose transporters GLUT-1 to -5 and SGLT-1 in the endothelium of rat coronary artery (Gaudreault et al. 2004, Diabetologica, 47, 2081-2092). In the present study the authors investigate and compare the presence and subcellular distribution of the classic glucose transporter isoforms in endothelial cells of cerebral, renal, and mesenteric arteries. The GLUTs and SGLT-1 were examined with immunohistochemistry and wide-field fluorescence microscopy coupled to deconvolution in en face preparation of intact artery. We identified GLUT-1 to -5 and SGLT-1 in the endothelial cells of all three vascular beds. The relative level of expression for each isoform was found comparable amongst arteries. Clusters of the glucose transporter isoforms were found at a high density in proximity to the cell-to-cell junctions. In addition, a consistent asymmetric distribution of GLUT-1 to -5 was found, predominantly located on the abluminal side of the endothelium in all three vascular beds examined (ranging from 68% to 91%, p<.05). The authors conclude that the expression and subcellular distribution of glucose transporters are similar in endothelial cells from vascular beds of comparable diameter and suggest that their subcellular organization may facilitate transendothelial transport of glucose in small contractile arteries.

Tuning properties of long period gratings in photonic bandgap fibers.

We investigate the thermal tuning properties of long period gratings (LPGs) in a fluid-filled photonic bandgap fiber (PBGF). The combination of strong, resonant waveguide dispersion, characteristic of all PBGF modes, and the large thermo-optic coefficients of fluids yields highly tunable grating resonances. We measure grating resonances in three transmission bands with large tuning coefficients of up to -1.58 nm/degrees C, which match numerical results. We derive an analytic model for the PBGF LPG tuning coefficient to show how it depends on both the shift of the transmission bands and the dispersion of the coupled modes.

Long period grating resonances in photonic bandgap fiber.

We demonstrate the formation of stress-induced long period gratings (LPGs) in fluid-filled photonic bandgap fiber (PBGF). Based on our experimental results, simulations, and theoretical understanding of LPGs, we identify coupling to a guided LP(11)-like mode of the core and lossy LP1x-like modes of cladding microstructure for a single grating period. The periodic modal properties of PBGFs allow for coupling to the same mode at multiple wavelengths without a dispersion turning point. Simulations identify inherent differences in the modal structure of even and odd bands.

Characterisation of glucose transporters in the intact coronary artery endothelium in rats: GLUT-2 upregulated by long-term hyperglycaemia.

AIMS/HYPOTHESIS: We have examined the effects of streptozotocin-induced type 1 diabetes on the expression and subcellular distribution of the classic sugar transporters (GLUT-1 to 5 and sodium-dependent glucose transporter-1 [SGLT-1]) in the endothelial cells of an en face preparation of septal coronary artery from Wistar rats. METHODS: The presence of the GLUT isoforms and SGLT-1 in the endothelial cell layer was determined by immunohistochemistry using wide-field fluorescence microscopy coupled to deconvolution, and was quantified by digital image analysis. RESULTS: We found that all of the transporters were expressed within these cells and that all except SGLT-1 were preferentially located on the abluminal side. The heaviest labelling was adjacent to the cell-to-cell junctions where the luminal and abluminal membranes are in close proximity, which may reflect a spatial organisation specialised for vectorial glucose transport across the thinnest part of the cytoplasm. Long-term hyperglycaemia, induced by streptozotocin, significantly downregulated GLUT-1, 3, 4 and 5 and dramatically upregulated GLUT-2, leaving SGLT-1 unchanged. CONCLUSIONS/INTERPRETATION: We conclude that the high susceptibility of endothelial cells to glucose toxicity may be the result of the subcellular organisation of their GLUTs and the increased expression of GLUT-2.

Pressure-dependent myogenic constriction of cerebral arteries occurs independently of voltage-dependent activation.

Pressure-induced decreases in arterial diameter are accompanied by membrane depolarization and Ca(2+) entry via voltage-gated Ca(2+) channels. Recent evidence also suggests the involvement of Ca(2+) sensitization of the contractile proteins. Both PKC and Rho kinase are candidate second messengers for the mediation of the sensitization process. We investigated the signaling pathways of pressure-induced decreases in rat cerebral artery diameter in vessels that were depolarized with a 60 mM potassium-physiological salt solution (KPSS). Arteries were mounted on a pressure myograph, and pressure-induced constrictions were recorded. In some experiments simultaneous changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) were recorded by using fura 2 fluorescence photometry. Pressure increases induced constriction with significant changes in [Ca(2+)](i) at high pressures (60-100 mmHg). The ratio of the change in diameter to change in [Ca(2+)](i) was greater for pressure-induced constriction compared with constriction produced by depolarization with 60 mM KPSS, suggesting that in addition to increases in [Ca(2+)](i), enhanced myofilament Ca(2+) sensitivity occurs during pressure-induced decreases in arterial diameter. Depolarizing the membrane with 60 mM KPSS increased [Ca(2+)](i) via a Ca(2+) influx pathway insensitive to PKC inhibition. Cerebral arteries were able to maintain their diameters in the continued presence of 60 mM KPSS. Pressure-induced constriction under these conditions was not associated with further increases in Ca(2+) but was abolished by selective inhibitors of PLC, PKC, and Rho kinase. We report for the first time that in rat cerebral arteries, pressure-induced decreases in arterial diameter are not only due to increases in voltage-gated Ca(2+) influx but also to accompanying increases in myofilament sensitivity to Ca(2+) mediated by PKC/Rho kinase activation.

Distribution of proteins implicated in excitation-contraction coupling in rat ventricular myocytes.

We have examined the distribution of ryanodine receptors, L-type Ca(2+) channels, calsequestrin, Na(+)/Ca(2+) exchangers, and voltage-gated Na(+) channels in adult rat ventricular myocytes. Enzymatically dissociated cells were fixed and dual-labeled with specific antibodies using standard immunocytochemistry protocols. Images were deconvolved to reverse the optical distortion produced by wide-field microscopes equipped with high numerical aperture objectives. Every image showed a well-ordered array of fluorescent spots, indicating that all of the proteins examined were distributed in discrete clusters throughout the cell. Mathematical analysis of the images revealed that dyads contained only ryanodine receptors, L-type Ca(2+) channels, and calsequestrin, and excluded Na(+)/Ca(2+) exchangers and voltage-gated Na(+) channels. The Na(+)/Ca(2+) exchanger and voltage-gated Na(+) channels were distributed largely within the t-tubules, on both transverse and axial elements, but were not co-localized. The t-tubule can therefore be subdivided into at least three structural domains; one of coupling (dyads), one containing the Na(+)/Ca(2+) exchanger, and one containing voltage-gated Na(+) channels. We conclude that if either the slip mode conductance of the Na(+) channel or the reverse mode of the Na(+)/Ca(2+) exchanger are to contribute to the contractile force, the fuzzy space must extend outside of the dyad.

Colocalization of dihydropyridine and ryanodine receptors in neonate rabbit heart using confocal microscopy.

Because of undeveloped T tubules and sparse sarcoplasmic reticulum, Ca(2+)-induced Ca(2+) release (CICR) may not be the major mechanism providing contractile Ca(2+) in the neonatal heart. Spatial association of dihydropyridine receptors (DHPRs) and ryanodine receptors (RyRs), a key factor for CICR, was examined in isolated neonatal rabbit ventricular myocytes aged 3-20 days by double-labeling immunofluorescence and confocal microscopy. We found a significant increase (P < 0.0005) in the degree of colocalization of DHPR and RyR during development. The number of voxels containing DHPR that also contained RyR in the 3-day-old group (62 +/- 1.8%) was significantly lower than in the other age groups (76 +/- 1.3 in 6-day old, 75 +/- 1.2 in 10-day old, and 79 +/- 0.9% in 20-day old). The number of voxels containing RyR that also contained DHPR was significantly higher in the 20-day-old group (17 +/- 0.5%) compared with the other age groups (10 +/- 0.7 in 3-day old, 11 +/- 0.6 in 6-day old, and 11 +/- 0.5% in 10-day old). During this period, the pattern of colocalization changed from mostly peripheral to mostly internal couplings. Our results provide a structural basis for the diminished prominence of CICR in neonatal heart.

alpha-actinin-2 couples to cardiac Kv1.5 channels, regulating current density and channel localization in HEK cells.

Voltage-gated K(+) (Kv) channels are particularly important in the physiology of excitable cells in the heart and the brain. PSD-95 is known to cluster Shaker channels and NMDA receptors and the latter is known to couple through alpha-actinin-2 to the post-synaptic cytoskeleton [Wyszynski et al. (1997) Nature 385, 439-442], but the mechanisms by which Kv channels are linked to the actin cytoskeleton and clustered at specific sites in the heart are unknown. Here we provide evidence that Kv1.5 channels, widely expressed in the cardiovascular system, bind with alpha-actinin-2. Human Kv1.5 interacts via its N-terminus/core region and can be immunoprecipitated with alpha-actinin-2 both after in vitro translation and from HEK cells expressing both proteins. The ion channels and alpha-actinin-2 co-localize at the membrane in HEK cells, where disruption of the actin cytoskeleton and antisense constructs to alpha-actinin-2 modulate the ion and gating current density.

Uptake of oxidized LDL by macrophages differs from that of acetyl LDL and leads to expansion of an acidic endolysosomal compartment.

Accumulation of cholesterol by macrophage foam cells in atherosclerotic lesions is thought to involve the uptake of modified low density lipoproteins (LDLs). Previous studies have shown that there is impaired degradation of oxidized LDL in macrophages. The present study was done to determine whether the differences in intracellular metabolism of oxidized LDL and acetyl LDL were associated with delivery to different intracellular compartments. Mouse peritoneal macrophages were incubated with 1,1'-dioctadecyl-3, 3,3',3'-tetramethylindocarbocyanine perchlo- rate-labeled oxidized LDL or 3,3'-dioctadecyloxacarbocyanine perchlorate-labeled acetyl LDL and examined by fluorescence microscopy. Deconvolution image analysis showed <10% colocalization of the 2 lipoproteins at incubation times ranging from 30 minutes to 6 hours. Subcellular fractionation of macrophages after incubation with (99m)Tc-labeled oxidized LDL revealed accumulation of the tracer in a compartment with a d=1.042 g/mL, consistent with endosomes. Surprisingly, there was a concurrent dramatic shift of the density of lysosomal marker enzymes from d=1.1 g/mL to the same fractions that contained (99m)Tc, indicating that this compartment was formed after fusion with primary lysosomes. Parallel experiments in J774 cells, a murine macrophage-like cell line, did not show a similar density shift, perhaps because of the slower rate of accumulation of oxidized LDL by these cells. Fluorescence microscopy of macrophages labeled with a lysosomotropic dye revealed a marked expansion of the acidic compartment after exposure of cells to oxidized LDL. We conclude that oxidized LDL and acetyl LDL are internalized by morphologically distinct pathways. Furthermore, because of its impaired lysosomal degradation, oxidized LDL causes expansion of and a decrease in the density of the lysosomal compartment in macrophages.

The role of protein kinase C isozymes in bombesin-stimulated gastrin release from human antral gastrin cells.

Two of the most effective stimuli of gastrin release from human antral G cells are bombesin and phorbol esters. Both agonists result in activation of the protein kinase C family of isozymes, however, the exact contribution of protein kinase C to the resultant release of gastrin has been difficult to assess, possibly due to the presence of multiple protein kinase C isozymes in the G cells. The results of the present study demonstrated that the human antral G cells expressed 6 protein kinase C isozymes alpha, gamma, theta, epsilon, zeta, and mu. Of these protein kinase C, gamma and theta were translocated by stimulation of the cells by either 10 nM bombesin or 1 nM phorbol ester. Inhibition of protein kinase Cmu (localized to the Golgi complex) did not decrease bombesin-stimulated gastrin release indicating that this isozyme was not involved in the secretory process. The use of selective antagonists of the calcium-sensitive conventional protein kinase C subgroup resulted in an increase in bombesin-stimulated gastrin release and indicated that protein kinase Cgamma was involved in the desensitization of the bombesin response.

Distribution of active protein kinase C in smooth muscle.

To localize activated protein kinase C (PKC) in smooth muscle cells, an antibody directed to the catalytic site of the enzyme was used to assess PKC distribution by immunofluorescence techniques in gastric smooth muscle cells isolated from Bufo marinus. An antibody to vinculin was used to delineate the cell membrane. High-resolution three-dimensional images of immunofluorescence were obtained from a series of images collected through focus with a digital imaging microscope. Cells were untreated or treated with agents that increase PKC activity (10 microM carbachol for 1 min, 1 microM phorbol 12-myristate 13-acetate (PMA) for 10 min), or have no effect on PKC activity (1 micrometer 4-alpha phorbol, 12,13-didecanoate (4-alpha PMA)). In unstimulated cells, activated PKC and vinculin were located and organized at the cell surface. Cell cytosol labeling for activated PKC was sparse and diffuse and was absent for vinculin. After treatment with carbachol, which stimulates contraction and PKC activity, in addition to the membrane localization, the activated PKC exhibited a pronounced cytosolic fibrillar distribution and an increased total fluorescence intensity relative to vinculin. The distributions of activated PKC observed after PMA but not 4-alpha PMA were similar to those observed with carbachol. Our results indicate that in resting cells there is a pool of activated PKC near the cell membrane, and that after stimulation activated PKC is no longer membrane-confined, but is present throughout the cytosol. Active PKC appears to associate with contractile filaments, supporting a possible role in modulation of contraction.

Superresolution three-dimensional images of fluorescence in cells with minimal light exposure.

Fluorescent probes offer insight into the highly localized and rapid molecular events that underlie cell function. However, methods are required that can efficiently transform the limited signals from such probes into high-resolution images. An algorithm has now been developed that produces highly accurate images of fluorescent probe distribution inside cells with minimal light exposure and a conventional light microscope. This method provides resolution nearly four times greater than that currently available from any fluorescence microscope and was used to study several biological problems.

Comparison of lipid composition of milk from half-Danish Jersey cows and United States Jersey cows.

We studied differences in lipid composition of milk from Jersey cows with US sires and from Jersey cows with Danish sires. Milk samples were obtained on DHIA test day from 32 cows with Danish sires and 32 herdmates with US sires in two herds. The Jerseys with US sires were paired with those with Danish sires by parity and stage of lactation. Mean percentage of milk fat was 5.7%, for Jerseys with Danish sires and 4.8% for Jerseys with US sires. Total fat per day was the same (.91 kg) for both groups. Detailed analysis of milk lipids indicated that lipid composition of milk was similar for cows with US sires and those with Danish sires. However, milk from Jerseys with Danish sires contained more free cholesterol than milk from Jerseys with US sires, 17.5 versus 14.3 +/- .6 mg/dl. The proportion of polyunsaturated fatty acids was greater for milk from Jerseys with US sires than for milk from Jerseys with Danish sires (2.3 vs. 2.1%). Although lipid composition of milk from both groups was generally similar, the milk of Jersey cows with Danish sires had higher concentrations of free cholesterol and lower proportions of polyunsaturated fatty acids, both of which are possible negative factors for health of consumers.

Coupling of the Na+/Ca2+ exchanger, Na+/K+ pump and sarcoplasmic reticulum in smooth muscle.

The Na+/Ca2+ exchanger, driven by a transmembrane Na+ gradient, plays a key role in regulating Ca2+ concentration in many cells. Although the exchanger influences Ca2+ concentration, its activity in smooth muscle appears to be closely coupled to Ca2+ availability from intracellular stores. This linkage might result if the exchanger were positioned close to Ca2+ storage sites within the sarcoplasmic reticulum. To test this hypothesis we have developed methods to assess the relative three-dimensional distribution of proteins involved in Na+/K+ pumping, Na+/Ca2+ exchange, Ca2+ storage within the sarcoplasmic reticulum, and attachment of contractile filaments to the membrane in smooth muscle. Here we report that the Na+/Ca2+ exchanger is largely co-distributed with the Na+/K+ pump on unique regions of the plasma membrane in register with, and close to, calsequestrin-containing regions of the sarcoplasmic reticulum in sites distinct from the sites where contractile filaments attach to the membrane. This molecular organization suggests that the plasma membrane is divided into at least two functional domains, and appear to provide a mechanism for the strong linkage seen in smooth muscle between Na+/K+ pumping and Na+/Ca2+ exchange, and between Na+/Ca2+ exchange and Ca2+ release from the sarcoplasmic reticulum.

Isoproterenol stimulates rapid extrusion of sodium from isolated smooth muscle cells.

beta-Agonists cause an inhibition of contractility and a transient stimulation of Na+/K+ pumping in smooth muscle cells of the stomach from the toad Bufo marinus. To determine if the stimulation of Na+/K+ pumping causes changes in intracellular [Na+] ([Na+]i) that might link Na+ pump stimulation to decrease Ca2+ availability for contraction, [Na+]i was measured in these cells with SBFI, a Na(+)-sensitive fluorescent indicator. Basal [Na+]i was 12.8 +/- 4.2 mM (n = 32) and was uniform throughout the cell. In response to isoproterenol, [Na+]i decreased an average of 7.1 +/- 1.1 mM in 3 sec. Since this decrease in [Na+]i could be completely blocked by inhibition of the Na+ pump, or by blockade of the beta-receptor, [Na+]i reduction is the result of occupation of the beta-receptor by isoproterenol and subsequent stimulation of the Na+ pump. 8-Bromoadenosine 3',5'-cyclic monophosphate and forskolin mimicked the effect of isoproterenol, indicating that the sequence of events linking beta-receptor occupation to Na+ pump stimulation most likely includes activation of adenylate cyclase, production of cAMP, and stimulation of cAMP-dependent protein kinase. The decrease in [Na+]i is sufficiently large and fast that it is expected to stimulate turnover of the Na+/Ca2+ exchanger in the Ca2+ extrusion mode, thereby accounting for the observed linkage between stimulation of the Na+/K+ pump and inhibition of contractility in response to beta-adrenergic agonists.

Infrapopliteal angioplasty: long-term follow-up.

PURPOSE: The authors report their experience with 55 intrapopliteal percutaneous transluminal angioplasty (PTA) procedures performed primarily for limb salvage between May 1986 and June 1992. PATIENTS AND METHODS: Forty patients who underwent 55 PTA procedures were followed up prospectively. Multiple risk factors were present in 41 cases (75%). Twenty-three procedures were performed in patients with distal saphenous vein (n = 7) and femoropopliteal (n = 16 [13 were polytetrafluoroethylene]) bypass grafts. Follow-up was available for 54 of the 55 episodes. RESULTS: Technical success was achieved in 52 procedures (95%), with long-term clinical success in 23 of these 52 (44%) at an average follow-up of 25.8 months (range, 1-72 months). Failures tended to occur within the first 3 months. Of the eight PTA procedures in patients with distal saphenous vein bypass grafts, only two remained successes during the course of the study, at an average follow-up of only 2.5 months. Similarly, only two of 15 procedures performed in patients with femoropopliteal grafts remained successes. No complications resulted in emergency surgery or altered the original surgical reconstructive options. CONCLUSION: Infrapopliteal PTA is a safe and effective treatment for native vessel disease in selected patients facing surgical reconstruction for limb salvage but is less durable in patients who have undergone previous bypass procedures.

Correlated responses in reproduction accompanying selection for milk yield in Jerseys.

Reproductive traits of heifers and primiparous cows from a long-term selection project were analyzed to determine correlated response to single-trait selection for milk yield. Data were from 1056 daughters (765 selection, 291 control) of 37 bulls (17 selection, 20 control). Traits in heifers were ages at first observed estrus and at first breeding, services to conception, interval from first service to conception, and length of first gestation. Traits in primiparous cows were ages at first calving and at first breeding, after calving; services to conception; length of second gestation; and intervals from calving to first observed estrus, to first breeding, and to conception, from first service to conception, and from first to second calving. Analyses for services to conception in heifers and primiparous cows were categorical using models containing genetic group and generation. Analyses of other traits were by linear mixed models using fixed effects of genetic group, generation within group, and year-season of birth. Sires were assumed random and nested within genetic group. The mean square for sires within group was used to test for group differences. No significant differences were found between genetic groups in traits measured in heifers; however, the interval from first service to conception approached significance (control superior). In primiparous cows, differences between genetic groups were significant for the intervals of calving to first breeding and calving to conception and for length of second gestation (control superior). For other traits, reproductive performance of the control was better but not significantly different from that of the selected group. Reproductive performance should be monitored during selection for high milk yield.

Correlated response in growth and body measurements accompanying selection for milk yield in Jerseys.

Growth and body measurements from a long-term selection project were analyzed to determine correlated responses to single-trait selection for milk yield. Data were from 1056 daughters (765 selection, 291 control) of 37 bulls (17 selection, 20 control) of 37 bulls (17 selection, 20 control) and included BW and measures of heart girth, chest depth, wither height, and length from withers to pins and from withers to hooks taken at 6 mo, 15 mo, first calving, end of first lactation, and maturity. Other data were birth weight, change in measurements and weights from first calving to end of first lactation, monthly rate of gain from 1 to 13 mo of age, and age reaching breeding weight (250 kg). Principal component scores were calculated from standardized measurements at each age. The first three principal components has meaning (size, length vs. girth, and height vs. girth). All analyses used linear mixed models with fixed effects of genetic group, generation within group, year-season of birth or calving, parity of dam, and birth status (multiple or single birth). Sires were assumed to be random and nested within genetic group. Mean squares for sires was used to test for group differences. Generation did not differ in any analysis and was removed from all models. Selection cows were heavier, larger in some measurements, and had greater overall size at 6 mo of age. Selection cows had greater monthly rate of gain and attained breeding weight at an earlier age. Genetic groups did not differ for any other measurement or weight. Control cows gained more weight and increased more in some measurements between first calving and end of first lactation. Selection for milk yield did not result in an undesirable correlated response in an growth or body measurement.