Calcium Imaging and Caged Compounds

Roger Thompson

BSC 5936

Spring 2005

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

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

Instrumentation

• Detection system– Fluorophore

– Wavelength filters

– Detector

– Excitation source

• Types of instruments– Spectrofluorometer

– Fluorescence Microscope

– Flow cytometer

Chemical Fluorescent Indicators

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

• Indicator’s light intensity• Other physiological parameters

– Simultaneous patch-clamp

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

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

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

Bioluminescent Calcium Indicators

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

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

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

Dye-loading Procedures

• 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

• 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

• 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

• 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

• 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

Potential Problems of Ca2+ Indicators and their Solutions

• Intracellular buffering– Indicator can alter

[Ca2+]i when loaded in high concentration

• Cytotoxicity– Can damage some

types of cells

– Can affect redox metabolism or cell proliferation

• 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

• 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

• 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

Techniques for measuring Ca2+

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

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

Caged Compounds

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

Ratiometric intracellular calcium imaging in the isolated beating

rat heart using indo-1 fluorescence

Eerbeek etal., 2004

J. Appl. Physiol. 97:2042-2050.

Purpose

• Describes an optical ratio imaging setup and an analysis method for the beat-to-beat Cai

2+ 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.

Why?

• Because abnormalities in intracellular calcium (Cai

2+) 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.

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

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.

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 (Cai

2+) 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.

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.

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.

 

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.

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.

 

 

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).

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.

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.

Results

• Cai2+ transients show that Cai

2+ 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 Cai

2+ with a high temporal (1 ms) and spatial (0.6mm) resolution in the beating heart

Inhibition of inositol 1,4.5-trisphosphate-induced Ca2+

release by cAMP-dependent protein kinase in a living cell

Tertyshnikova & Fein, 1998

PNAS 95:1613-1617

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?

Ca2+ & cAMP

• Ubiquitous intracellular 2nd messengers

• Believed to modulate each other

• cAMP can potentiate or inhibit agonist-induced Ca2+ elevation depending on cell type

How tested?

• Used caged IP3, caged Ca2+, and caged cAMP to rapidly elevate their [C]i

• 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

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.