Gregor Zupančič

Differential exocytosis from human endothelial cells evoked by high intracellular Ca2+ concentration

Endothelial cells secrete a range of procoagulant, anticoagulant and inflammatory proteins by exocytosis to regulate blood clotting and local immune responses. The mechanisms regulating vesicular exocytosis were studied in human umbilical vein endothelial cells (HUVEC) with high‐resolution membrane capacitance (Cm) measurements. The total whole‐cell Cm and the amplitudes and times of discrete femtoFarad (fF)‐sized Cm steps due to exocytosis and endocytosis were monitored simultaneously. Intracellular calcium concentration [Ca2+]i was elevated by intracellular photolysis of calcium‐DM‐nitrophen to evoke secretion and monitored with the low‐affinity Ca2+ indicator furaptra. Sustained elevation of [Ca2+]i to > 20 μm evoked large, slow increases in Cm of up to 5 pF in 1‐2 min. Exocytotic and endocytotic steps of amplitude 0.5‐110 fF were resolved, and accounted on average for ≈33 % of the total Cm change. A prominent component of Cm steps of 2.5‐9.0 fF was seen and could be attributed to exocytosis of von‐Willebrand‐factor‐containing Weibel‐Palade bodies (WPb), based on the near‐identical distributions of capacitance step amplitudes, with calculated estimates of WPb capacitance from morphometry, and on the absence of 2.5‐9.0 fF Cm steps in cells deficient in WPb. WPb secretion was delayed on average by 23 s after [Ca2+]i elevation, whereas total Cm increased immediately due to the secretion of small, non‐WPb granules. The results show that following a large increase of [Ca2+]i, corresponding to strong stimulation, small vesicular components are immediately available for secretion, whereas the large WPb undergo exocytosis only after a delay. The presence of events of magnitude 9‐110 fF also provides evidence of compound secretion of WPb due to prior fusion of individual granules.

Temperature-Dependence of Weibel-Palade Body Exocytosis and Cell Surface Dispersal of von Willebrand Factor and Its Propolypeptide

Background Weibel-Palade bodies (WPB) are endothelial cell (EC) specific secretory organelles containing Von Willebrand factor (VWF). The temperature-dependence of Ca2+-driven WPB exocytosis is not known, although indirect evidence suggests that WPB exocytosis may occur at very low temperatures. Here we quantitatively analyse the temperature-dependence of Ca2+-driven WPB exocytosis and release of secreted VWF from the cell surface of ECs using fluorescence microscopy of cultured human ECs containing fluorescent WPBs. Principal Findings Ca2+-driven WPB exocytosis occurred at all temperatures studied (7–37°C). The kinetics and extent of WPB exocytosis were strongly temperature-dependent: Delays in exocytosis increased from 0.92 s at 37°C to 134.2 s at 7°C, the maximum rate of WPB fusion decreased from 10.0±2.2 s−1 (37°C) to 0.80±0.14 s−1 (7°C) and the fractional extent of degranulation of WPBs in each cell from 67±3% (37°C) to 3.6±1.3% (7°C). A discrepancy was found between the reduction in Ca2+-driven VWF secretion and WPB exocytosis at reduced temperature; at 17°C VWF secretion was reduced by 95% but WPB exocytosis by 75–80%. This discrepancy arises because VWF dispersal from sites of WPB exocytosis is largely prevented at low temperature. In contrast VWF-propolypeptide (proregion) dispersal from WPBs, although slowed, was complete within 60–120 s. Novel antibodies to the cleaved and processed proregion were characterised and used to show that secreted proregion more accurately reports the secretion of WPBs at sub-physiological temperatures than assay of VWF itself. Conclusions We report the first quantitative analysis of the temperature-dependence of WPB exocytosis. We provide evidence; by comparison of biochemical data for VWF or proregion secretion with direct analysis of WPB exocytosis at reduced temperature, that proregion is a more reliable marker for WPB exocytosis at reduced temperature, where VWF-EC adhesion is increased.

Light dependence of oxygen consumption by blowfly eyes recorded with a magnetic diver balance

We measured the oxygen (O2) consumption of isolated blowfly eyes using a magnetic diver balance, a device for high-resolution volumetric O2 consumption measurements. The light-induced O2 consumption is at most three times the value of the dark consumption, which is 0.6xa0nl O2xa0s−1xa0eye−1, and is in good agreement with the estimates based on electrophysiological data. With longer stimuli the increase follows a double exponential time course. The respective time constants are approximately 2 and 20xa0s and show no dependence on light intensity, whereas the dependence of amplitudes can be fitted by a Hill equation. Decreasing the stimulus duration reveals that the peak in O2 consumption overshoots the time course induced by long stimuli. We suggest this may be a general feature of mitochondrial activation. The dependence of the O2 consumption peak on stimulus duration at high light intensity has a hump with stimulus durations of 10–20xa0ms, coinciding with the stimulus durations that start to induce the adaptation of the receptor potential.

Human anterior lens capsule epithelial cells contraction

Purpose:u2002 Human anterior lens epithelial cells, attached to surgically isolated capsules, were found to contract upon stimulation. The purpose of this study was to characterize these contractions, which create gaps between cells, and to assess the underlying physiological mechanisms and their possible association with cataract formation.

Singing Greeting Card Beeper as a Finger Pulse Sensor.

We constructed a robust and low-priced finger pulse sensor from a singing greeting card beeper. The beeper outputs the plethysmographic signal, which is indistinguishable from that of commercial grade sensors. The sensor can be used in school for a number of experiments in human cardiovascular physiology.

The Effect of Gentian Violet on Human Anterior Lens Epithelial Cells

Abstract Purpose: To evaluate whether the gentian violet staining of the anterior lens capsule during the cataract surgery is cytotoxic for the human lens epithelial cells, as an indirect indication of possible toxicity towards the corneal endothelium and the safety of gentian violet application. Materials and methods: Two groups of anterior lens capsules obtained during the cataract surgery, gentian violet stained and non-stained, were incubated with fluorescent dye Fura-2. Their fluorescence, upon excitation at 360 and 380u2009nm, was imaged to monitor changes in free intracellular calcium concentration ([Ca2+]i) in response to pharmacological stimulation by acetylcholine. The [Ca2+]i homeostasis is the indicator of cellular function. The changes in [Ca2+]i were compared between the two groups. Results: Epithelial cells responded to acetylcholine in both groups of capsules - gentian violet stained (nu2009=u200917) and non-stained ones (nu2009=u200933). No significant differences of the elicited responses were found in rise time (pu2009=u20090.89), decay time (pu2009=u20090.61) or amplitude of [Ca2+]i (pu2009=u20090.96 for 63× and pu2009=u20090.26 for 40× objectives) between the two groups of capsules (Student t test). Conclusions: The staining of the anterior lens capsule with gentian violet during phacoemulsification in concentration of 0.01%, does not have detectable cytotoxic effects, which would affect the [Ca2+]i homeostasis in lens epithelial cells. The data, if extrapolated to corneal endothelium, exposed to the same concentration, suggest that gentian violet in concentration of 0.01% is safe as an adjunct for capsule visualization in cataract surgery.

A method for dynamic spectrophotometric measurements in vivo using principal component analysis-based spectral deconvolution

A method was developed for dynamic spectrophotometric measurements in vivo in the presence of non-specific spectral changes due to external disturbances. This method was used to measure changes in mitochondrial respiratory pigment redox states in photoreceptor cells of live, white-eyed mutants of the blowfly Calliphora vicina. The changes were brought about by exchanging the atmosphere around an immobilised animal from air to N2 and back again by a rapid gas exchange system. During an experiment reflectance spectra were measured by a linear CCD array spectrophotometer. This method involves the pre-processing steps of difference spectra calculation and digital filtering in one and two dimensions. These were followed by time-domain principal component analysis (PCA). PCA yielded seven significant time domain principal component vectors and seven corresponding spectral score vectors. In addition, through PCA we also obtained a time course of changes common to all wavelengths—the residual vector, corresponding to non-specific spectral changes due to preparation movement or mitochondrial swelling. In the final step the redox state time courses were obtained by fitting linear combinations of respiratory pigment difference spectra to each of the seven score vectors. The resulting matrix of factors was then multiplied by the matrix of seven principal component vectors to yield the time courses of respiratory pigment redox states. The method can be used, with minor modifications, in many cases of time-resolved optical measurements of multiple overlapping spectral components, especially in situations where non-specific external influences cannot be disregarded.

Development and plasticity of mitochondria and electrical properties of the cell membrane in blowfly photoreceptors

Blowfly photoreceptors are highly energy demanding sensory systems. Their information processing efficiency is enabled by the high temporal resolution of the cell membrane, requiring heavy metabolic support by the mitochondria. We studied the developmental changes of the mitochondrial apparatus and electrical properties of the photoreceptor membrane in the white eyed Calliphora vicina Chalky. Using in vivo microspectrophotometry and Western blot analysis, we found an age-dependent increase in the concentration of mitochondrial pigments. The maximal change occurred during the first week. The age-related changes were smaller in dark-bred than in light-bred flies. The mitochondrial pigment content increased after the switch from dark to light rearing and decreased after the switch from light to dark rearing. The electrical parameters of the photoreceptors were investigated with intracellular recordings. The resting membrane resistance and time constant decreased significantly after eclosion. The decrease was again most significant during the first week of adult life, paralleled with changes in the Na/K pump-dependent hyperpolarizing afterpotential. We conclude that the photoreceptor mitochondria exhibit remarkable ontogenetic and phenotypic plasticity, because the quantity of mitochondrial pigments tightly follows the development of the cell membrane as well as the energy demands of the photoreceptors under different rearing conditions.

Changes in redox states of respiratory pigments recorded from the eyes of live blowflies exposed to light stimuli and hypoxia

Time courses of mitochondrial responses to illumination-induced physiological loads and to hypoxia, were recorded optically from eyes of blowflies Calliphora vicina chalky. We isolated changes in redox states of haems a3, a, c, and b. Two types of responses to light stimulation were observed. Haems b and a3 responded with transient oxidation and haems a and c with reduction. The same two groups emerged in response to anoxic exposure. The onset of reduction of haems a and c had virtually no latency, while haems a3 and b exhibited a transient oxidation followed by reduction only after 10–20xa0s. The dependence of the steady-state reduction level on