Calcium Imaging and Caged Compounds

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Calcium Imaging and Caged Compounds

• Roger Thompson
• BSC 5936
• Spring 2005

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Introduction

• Calcium cannot be visualized directly
• Specific molecules are used that have optical
  properties which change upon interacting with
  calcium
• Calcium concentrations can change in
  milli-seconds
• Calcium acts as a universal 2nd messenger
• Calcium regulates cellular processes such as
  muscle contraction, fertilization, cell division,
  blood clotting, and synaptic transmission

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Fluorescence

• Requires molecules called fluorophores or
  fluorescent dyes
• Properties include absorbance, lifetime,
  intensity, spectra
• Is the result of a three stage process
• 3 stages are
• Excitation
• Excited-state lifetime
• Fluorescence emission

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Instrumentation

• Detection system
• Fluorophore
• Wavelength filters
• Detector
• Excitation source
• Types of instruments
• Spectrofluorometer
• Fluorescence Microscope
• Flow cytometer

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Chemical Fluorescent Indicators
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Selection Criteria

• Calcium Concentration range
• Near dissociation constant (Kd) (best)
• Detectable at 0.1Kd to 10Kd
• Delivery method
• Measurement
• Quantitative or qualitative
• Ion concentration
• Instruments
• Sources of noise
• Indicators light intensity
• Other physiological parameters
• Simultaneous patch-clamp

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Ultraviolet Wavelength Excitation Fluorescent
Indicators

• Fura-2, Indo-1 and derivatives
• Quin-2 and derivatives
• Intermediate Ca2-binding affinity
• Fura-4F, Fura-5F Fura-6F plus
• Benzothiaza-1 2
• Low-affinity indicators
• Fura-FF, BTC, Mag-Fura-2, Mag-Fura-5, and
  Mag-Indo-1

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Visible Wavelength Excitation Fluorescent
Indicators

• Fluo-3, Rhod-2 and derivatives
• Low-affinity indicators
• Fluo-5N, Rhod-5N, X-Rhod-5N and derivatives
• Calcium Green, Calcium Orange, Calcium Crimson
• Oregon Green 488 BAPTA indicators
• Fura Red indicator
• Calcein

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Ratiometric vs. NonRatiometric

• Ratiometric
• Includes indo-1 and fura-2
• Allows for correction of differences of path
  length and accessible volume in three dimensional
  specimens
• Indicator has 2 excitable wavelengths
• Indo-1 at 405- and 485 nm
• Nonratiometric
• Includes fluo-3, rhod-2 and the calcium green
  class

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Bioluminescent Calcium Indicators
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Bioluminescence

• Light produced by biological organisms
• Low intensity
• Assays are sensitive and free of background
• One example is Aequorin
• Photoprotein isolated from luminescent jellyfish
• Is not exported or secreted not
  compartmentalized
• Usually microinjected into cells
• Has 3 Ca2 binding sites

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Ca2-binding Photoproteins

• Visible bioluminescence by an intramolecular
  reaction in the presence of calcium
• Simple instrument requirements
• Not affected by photobleaching
• Ex Obelin and Aequorin
• Problems
• Loading, detection and calibration

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Green Fluorescent Protein-based

• Photosensitive proteins synthesized by the
  jellyfish Aequorea victoria
• Can be cloned and fused with DNA
• Provides marker for gene expression and protein
  localization in living organisms

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Dye-loading Procedures
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• Ester Loading
• Derivatized with an AM (acetoxymethyl) ester
• Passively diffuses through plasma membrane
• Subject to compartmentalization or incomplete
  hydrolysis

• Microinjection
• Intermittent injection of indicator dissolved in
  cytosolic-like solution via glass micropipette
• Invasive technique

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• Diffusion from Patch-Clamp pipettes
• Passive microinjection i.e. perforated patch
  technique using substance such as Nystatin

• Diffusion through Gap Junctions
• Retrograde perfusion w/ a low Ca2 solution
  containing collagenase and proteases followed by
  mechanical dissociation of cells

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• ATP-induced permeabilization
• Extracellular ATP induces cation flux and
  increases permeability of the plasma membrane via
  ATP4- receptor

• Hyposmotic Shock Treatment
• Several washes in Ca2 free solution, followed by
  hyposmotic solution containing indicator
• Can kill cells

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• Gravity Loading
• Ca2 free solution wash
• Centrifugation
• Incubation with indicator
• Centrifugation

• Scrape Loading
• Cultured cells are scraped from dish while in
  buffer containing indicator
• Scraping rips holes in membrane

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• Lipotransfer Delivery Method
• Uses membrane permeant cationic liposomes
  containing indicator/dye

• Fused Cell Hybrids
• Photoproteins are loaded by fusing cells and
  human erythrocyte ghosts contain the
  photoprotein in a medium containing Sendai virus

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Potential Problems of Ca2 Indicators and their
Solutions
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• Intracellular buffering
• Indicator can alter Ca2i when loaded in high
  concentration

• Cytotoxicity
• Can damage some types of cells
• Can affect redox metabolism or cell proliferation

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• Autofluorescence
• Collagen fibers and calcifications can give off
  autofluorescence
• Pyridine nucleotides may also. These include
  NADH, NADP, FAD, and FMN

• Bleaching
• Too much illumination or photodamage
• Can be diminished by adding O2 or an antioxidant

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• Compartmentalization
• Indicator becomes trapped within some
  intracellular organelles and is not homogeneous
  throughout the cell

• Binding to Other Ions Proteins
• Indicators may bind to intracellular proteins and
  alter their fluorescent properties including
  changes in spectrum, kinetics, or Kd

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• Dye Leakage
• Indicators may leak from the cytosol into the
  extracellular medium
• Is regulated by anion transporter system
• Can be inhibited by probenecid, sulfinpyrazone
  or low temperature

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Techniques for measuring Ca2
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Optical Techniques

• Multiparameter digitized video microscopy
• Confocal laser scanning microscopy
• Scans specimen collecting emitted fluorescence
  thru a pinhole
• Two-photon excitation laser scanning microscopy
• One fluorophore is excited by two individual
  photons simultaneously
• Pulsed-laser imaging for rapid Ca2 gradients
• Allows submicron spatial resolution and msec
  temporal resolution
• Time-resolved fluorescence lifetime imaging
  microscopy (FLIM)
• ( ? ) time in excited state to ground state
• Photomultiplier tube
• Flow cytometry
• Flowing stream suspension

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Non-Optical Techniques

• Electrophysiology
• Currents generated by Ca2-dependent ion channels
• Ca2-selective electrodes
• Ion-complexing ligand in a liquid lipophilic
  membrane
• Vibrating Ca2-selective probe
• Ion-selective non-invasive located w/in microns
  of the cell surface measures ion flux

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Caged Compounds
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Characteristics

• Photosensitive chelators of ions or substances
  such as Ca2, H, ATP, or IP3
• Exist as an inactive form due to combination
  with radicals such as the nitrophenyl group
• chemically caged
• Activated by exposure to UV illumination
• Release of Ca2 in ?sec or msec

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Ratiometric intracellular calcium imaging in the
isolated beating rat heart using indo-1
fluorescence

• Eerbeek etal., 2004
• J. Appl. Physiol. 972042-2050.

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Purpose

• Describes an optical ratio imaging setup and an
  analysis method for the beat-to-beat Cai2
  videofluorescence images of an indo-1 loaded,
  isolated Tyrode-perfused beating rat heart.
• Possible to register different temporal Cai2
  transients together with left ventricle pressure
  changes.

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Why?

• Because abnormalities in intracellular calcium
  (Cai2) handling has been implicated as the
  underlying mechanism in a large number of
  pathologies in the heart i.e., contraction,
  electrophysiological properties, mitochondrial
  function, heart failure, and cardiac hypertrophy.

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How?

• Used indo-1. Requires a single-excitation
  wavelength of light and two emission wavelengths
  to be recorded
• Visualization of Cai2 changes in time and space
• A multiviewer (filters/dichroic mirrors) split
  emission light into two wavelengths projected
  the two mages on a CCD chip of a CCD camera

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Fig. 1. A schematic diagram of the
dual-wavelength videofluorometric measurement
system. The 365-nm excitation light, which is
provided by a 100-W Mercury (Hg) arc lamp, is
selected by a 365-nm filter (UG-1 filter), DCLP
390 is a dichroic mirror in the beam splitter,
which is translucent for wavelengths higher than
385 nm. B the dichroic mirrors DCLP 455 are
translucent for wavelengths higher than 455 nm.
Both light paths use filters 405_ 10nm and 485
_ 20 nm, respectively. Both images are
projected simultaneously on the cathode (I) of a
second-generation image intensifier tube by a
macro lens charge-coupled device (CCD) of a
cooled video camera. BP, band-pass filter LVP,
left ventricular pressure.
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Fig. 2. A a hear image is shown on which the 5
X 5 matrix pattern (spatial resolution 1.8 mm) is
superimposed to illustrate how the spatial
dependency of the cytosolic calcium (Cai2) was
analyzed. The whole matrix, including the
uppermost left part, is covering the left
ventricle of the heart. Symbols are the same as
Fig. 6 (symbols are associated with that heart
position). RP, reference point for the image
processing. B the three traces show the indo-1
ratio signals of the 3 selected areas during a
whole heart cycle before filtering.
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Fig. 3. NADH autofluorscence (NADH / uranyl)
measured at 450 nm, during the first 100 ms of
the heart cycle, before and after a mimicked
loading procedure.
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Fig 4. Two images are shown at 485 nm. A NADH
image (487nm). B the same heart after loading
with indo-1 at the same wavelength.  
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Fig. 5. Image at both wavelengths (405 and 485
nm) and the ratio image after a small lesion
(pinching the tissue) on the epicardium of an
indo-1-loaded heart.
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Fig. 6. Time course of the indo-1 ratio (?) and
the left ventricular pressure (0) during a whole
heart cycle of 200 ms (pacing frequency 300/min).
The indo-1 ratio is corrected for the
autofluorescence.    
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Fig. 7. Time course of the indo-1 ratio of the 3
squares o, , and ? (positions shown in Fig. 2A)
produced with a 5 X 5 matrix (spatial resolution
1.8 mm) during a heart cycle of 200 ms (pacing
frequency 300/min). A and B with stimulation of
the right atrium, and C and D, with direct
stimulation of the left ventricle, show the time
course of the indo-1 ratio. B and D after
addition of 2,3-butanedione monoxine (BDM) (
diacetyl monoxine DAM). Inset in A and C shows
the schematic heart with the position of the 3
squares in the matrix and in C also the position
of the stimulation electrode (stim).
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Fig. 8. A and B Time course of the indo-1
ratio at different locations in 2 different
hearts (positions shown in each matrix) produced
with a 15 X 15 matrix (spatial resolution 0.6 mm)
during the first 100 ms of the heart cycle
(pacing frequency 300/min). In the horizontal
direction a delay in calcium transients is
present from left to right.
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Fig. 9. A steps taken from the acquired image
from the CCD to a detailed analysis of calcium
transients in a small portion of the left
ventricle. The splitting of the CCD output into
2 images, representing the 405- and 485-nm
wavelengths, is performed by using the 2
reference points (see MATERIALS AND METHODS).
The contrast and brightness of the ratio image
(image at 405 nm divided by the image at 485 nm)
is optimized to show more clearly the region of
interest. It is clear that the ratio method is
effective in canceling out inhomogeneities in the
fluorescence at the same wavelengths. The area
of interest is 4.2 by 4.2 mm and was analyzed by
dividing it in 7 parts in either the horizontal
or vertical direction. B results of the
analysis in both the horizontal (vertical binned)
and vertical (horizontal binned) direction from 1
heart. In the horizontal direction, a delay in
calcium transients is present from the left (1)
to the right (7). This delay is absent in the
vertical direction.
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Results

• Cai2 transients show that Cai2 activation
  propagates horizontally from the left to right
  during sinus rhythm or from the stimulus site
  during direct left ventricle stimulation.
• The indo-1 ratiometric video technique allows the
  imaging of ratio changes of Cai2 with a high
  temporal (1 ms) and spatial (0.6mm) resolution in
  the beating heart

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Inhibition of inositol 1,4.5-trisphosphate-induced
Ca2 release by cAMP-dependent protein kinase in
a living cell

• Tertyshnikova Fein, 1998
• PNAS 951613-1617

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Hypothesis

• Is the principal mechanism of cAMP-dependent
  inhibition of Ca2 mobilization by inhibition of
  IP3-induced Ca2 release or by stimulation of
  Ca2 removal from the cytoplasm?

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Ca2 cAMP

• Ubiquitous intracellular 2nd messengers
• Believed to modulate each other
• cAMP can potentiate or inhibit agonist-induced
  Ca2 elevation depending on cell type

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How tested?

• Used caged IP3, caged Ca2, and caged cAMP to
  rapidly elevate their Ci
• Carbacyclin elevates cAMP (via Gs
  protein-dependent activation of adenylyl cyclase)
  inhibits IP3
• KT5720 and IP20 used as inhibitors of carbacyclin
• SNP (sodium nitoprusside) inhibits cGMP

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Results

• Elevation of cAMP inhibits IP3-induced Ca2
  release in an intact cell but does not affect the
  removal of Ca2 from the cytoplasm.
• Inhibition is mediated by cAMP Protein Kinase.