Background: Photolabile chelators that release Ca
2+ upon illumination have been used extensively to dissect the role of this important second messenger in cellular processes such as muscle ...contraction and synaptic transmission. The caged calcium chelators that are presently available are often limited by their inadequate changes in Ca
2+ affinity, selectivity for Ca
2+ over Mg
2+ and sensitivity to light. As these chelators are all based on nitrobenzyl photochemistry, we explored the use of other photosensitive moieties to generate a new caged calcium with improved properties.
Results: Azid-1 is a novel caged calcium in which a fluorescent Ca
2+ indicator, fura-2, has been modified with an azide substituent on the benzofuran 3-position. Azid-1 binds Ca
2+ with a dissociation constant (K
d) of ∼230 nM, which changes to 120 μM after photolysis with ultraviolet light (330–380 nm). Mg
2+ binding is weak (8–9 mM K
d) before or after photolysis. Azid-1 photolyzes with unit quantum efficiency, making it 40–170-fold more sensitive to light than caged calciums used previously. The photolysis of azid-1 probably releases N
2 to form a nitrenium ion that adds water to yield an amidoxime cation; the electron-with-drawing ability of the amidoxime cation reduces the chelator's Ca
2+ affinity within at most 2 ms following a light flash. The ability of azid-1 to function as a caged calcium in living cells was demonstrated in cerebellar Purkinje cells, in which Ca
2+ photolytically released from azid-1 could replace the normal depolarization-induced Ca
2+ transient in triggering synaptic plasticity.
Conclusions: Azid-1 promises to be a useful tool for generating highly controlled spatial and temporal increases of Ca
2+ in studies of the many Ca
2+-dependent biological processes. Unlike other caged calciums, azid-1 has a substantial cross section or shows a high susceptibility for two-photon photolysis, the only technique that confines the photochemistry to a focal spot that is localized in three dimensions. Azide photolysis could be a useful and more photosensitive alternative to nitrobenzyl photochemistry.
Direct measurements of free Ca2+ in heart cells are needed for an understanding of the regulation of contractility. We developed and used Ca2+ -sensitive microelectrodes with fine tips, stable ...properties and ample sensitivity to free Ca2+ in the sub-micromolar range. In quiescent ventricular muscle, measurements which passed tests for electrode sealing and cell viability gave a mean free Ca2+ concentration of 0.26 microM. During contractures, we recorded Ca2+ transients rising as high as 10 microM. In studying the effects of catecholamines on free Ca2+ and force, we found evidence that adrenaline can reduce myofibrillar Ca2+ sensitivity in intact heart muscle.
The relationship between the free cytoplasmic Ca2+concentration, Ca2+i, and neurotransmitter release was investigated in guinea pig brain synaptosomes and the neurosecretory cell line PC12. Release ...was induced by α -latrotoxin, which acts in both Ca2+-containing and Ca2+-free incubation media, or by the classical depolarizing agents high K+and veratridine, which require extracellular Ca2+. Two complementary approaches were used to reveal changes of Ca2+i: (i) direct measurement by a fluorescent Ca2+indicator (quin2) and (ii) study of the Ca2+-dependent phosphorylation of a protein, synapsin I, located at the cytoplasmic surface of synaptic vesicles. Depolarizing agents, when applied in Ca2+-containing medium, induced the Ca2+ito increase promptly 3- to 6-fold, drastically increased synapsin I phosphorylation, and caused stimulation of transmitter release. With α -latrotoxin, the Ca2+iincrease was delayed and occurred at a slower rate, the increase of synapsin I phosphorylation was less drastic, and the release response was much more pronounced. In Ca2+-free medium, depolarizing agents released no transmitter and had no effect on Ca2+ior synapsin I phosphorylation, whereas with α -latrotoxin these processes were dissociated: considerable stimulation of the release without apparent change of Ca2+iand synapsin I phosphorylation. We conclude that the relationship between average Ca2+iand transmitter release is not straightforward and, in particular, that the release evoked by α -latrotoxin in Ca2+-free medium is mediated by a factor(s) other than bulk redistribution of Ca2+from intracellular stores.
Quantitative analysis of volumetric TPLSM data Ifarraguerri, A.; Thompson, B.; Tsai, P.S. ...
2004 2nd IEEE International Symposium on Biomedical Imaging: Nano to Macro (IEEE Cat No. 04EX821),
2004
Conference Proceeding
This paper describes initial algorithm development in support of research on a new all-optical histology technique based on two-photon laser scanning microscopy (TPLSM) and laser tissue ablation. ...This new imaging modality makes it possible to obtain quantitative information that can be used to measure physiological parameters of interest. We present our initial image processing approach and volumetric rendering results. Two examples of useful quantitative parameters are presented: measurement of blood vessel volume fraction and cell counting.
We use femtosecond laser pulses to serially image and ablate neuronal tissue. This allows histological imaging throughout the entire depth of the brain; a procedure that is unattainable by ...multiphoton imaging alone.
Heat shock caused significant changes in intracellular pH (pH
i
) and intracellular free calcium concentration ( Ca
2+
i) which occurred rapidly after temperature elevation. pH
i
fell from a resting ...level value at 25 degrees C of 7.38 +/- 0.02 (mean +/- standard error of the mean, n = 15) to 6.91 +/- 0.11 (n = 7) at 35 degrees C. The resting level value of Ca2+i in single Drosophila melanogaster larval salivary gland cells was 198 +/- 31 nM (n = 4). It increased approximately 10-fold, to 1,870 +/- 770 nM (n = 4), during a heat shock. When salivary glands were incubated in calcium-free, ethylene glycol-bis(beta-aminoethyl ether)-N,N′,N′-tetraacetic acid (EGTA)-buffered medium, the resting level value of Ca
2+
i was reduced to 80 +/- 7 nM (n = 3), and heat shock resulted in a fourfold increase in Ca
2+
i to 353 +/- 90 nM (n = 3). The intracellular free-ion concentrations of Na
+
, K
+
, Cl
-
, and Mg
2+
were 9.6 +/- 0.8, 101.9 +/- 1.7, 36 +/- 1.5, and 2.4 +/- 0.2 mM, respectively, and remained essentially unchanged during a heat shock. Procedures were devised to mimic or block the effects of heat shock on pHi and Ca
2+
i and to assess their role in the induction of heat shock proteins. We report here that the changes in Ca
2+
i and pHi which occur during heat shock are not sufficient, nor are they required, for a complete induction of the heat shock response.
Background: Fluorescent indicators that show alterations in excitation and/or emission spectra in response to changes in Ca
2+ are widely used for quantitative cytosolic Ca
2+ measurements. There are ...several reports of changes in apparent Ca
2+ due only to illumination, however. These results have been attributed either to photodamage to the cells or to photodegradation of the indicator. Light-induced alteration in the behavior of the dye or cells would severely hamper the interpretation of experimental data. We examined this phenomenon in indo-1 loaded cells using confocal laser scanning microscopy.
Results: Illumination of indo-1 loaded GH3 cells leads to a decrease in apparent basal Ca
2+ and decreased peaks after depolarization with KCI. When cells were double loaded with indo-1 and fluo-3, the effect of UV illumination was noticed only with the former dye. UV irradiation of indo-1 in simple buffers caused overall photobleaching and conversion to a fluorescent but Ca
2+-insensitive species. The latter effect cannot be canceled by ratiometric calibration and is due to loss of carboxymethyl groups from the anilino nitrogens. This photodegradation was inhibited by extracellular administration of 10 to 100 μM Trolox, a water-soluble vitamin E analog.
Conclusions: Photodegradation processes like that observed for indo-1 are likely to be possible for all cation indicators that contain bis(carboxymethyl)anilino moieties, which include essentially all fluorescent indicators for Ca
2+ and Mg
2+ currently in biological use. If unrecognized, this photochemical dealkylation leads to an underestimation of the analyte concentrations depending on the intensity and duration of illumination. The problem can be avoided by including cell-permeant antioxidants such as Trolox in the bathing solution. The ultimate solution would be to redesign the indicators to minimize photodegradation in the absence of antioxidants.
This chapter discusses the optical probes for cyclic 3',5'-adenosine monophosphate (cAMP), which was the first molecule to be explicitly recognized as a “second messenger,” a mediator between ...extracellular stimuli and intracellular biochemical responses. Many roles have been assigned to cAMP. However, one area where cAMP research has been lagging is the detailed exploration of the spatial and temporal dynamics of cAMP signaling. Optical probes for Ca2+ have revealed that Ca2+ signaling is often remarkably complex in fine structures. The chapter discusses various methods for measuring cAMP—namely, assay in cell lysates, immunocytochemistry, fluorescent analogues of cyclic nucleotides, and fluorescently labeled cAMP receptor protein from E. coli. The chapter also describes a major sample application, which is the visualization of the spread of cAMP within MA-10 Leydig tumor cells from sites of relatively localized hormone stimulation.
The accurate measurement of ionized intracellular calcium Ca++i in single cells by flow cytometry with the use of a new fluorescent calcium chelator, indo‐1, is described. We have developed a ...dependable in situ calibration technique that indicates a resting Ca++i in lymphocytes of 100 nM. The enhanced fluorescence of this probe permits its use at sufficiently low cytoplasmic concentrations that buffering of Ca++i transients does not occur.
The Ca++i response of small resting B lymphocytes to crosslinking of surface antigen receptors by anti‐immunoglobulin is heterogeneous. With maximal stimulus, the peak Ca++i response occurs in 10 to 20 seconds with most cells reaching levels >/1 μM. Mean Ca++i falls to between 300 and 800 nM by 100 seconds where it remains for more than 10 min. Anti‐δ is a more potent stimulus of increased Ca++i than anti‐μ in terms of both Ca++i level and fraction of B cells responding. Whether this is due to the greater density of surface IgD than IgM, a difference in signal transduction efficiency, or both, is not yet known. Surface immunoglobulin receptors are present in great excess. Less than 3% of surface immunoglobulin is crosslinked at the peak of the Ca++i response.
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