calcium measurements in mitos

Richard Kondo kondo at buphy.bu.edu
Wed Aug 9 08:04:19 EST 1995


John Arne R|ttingen (j.a.rottingen at basalmed.uio.no) wrote:
: In article <3uq1uv$5fr at surz03.HRZ.Uni-Marburg.DE>, klieber at papin.HRZ.Uni-Marburg.DE (Hans-Georg Klieber) says:
: >
: >We woud like to measure the calcium content of mitochondria
: >in intact heart muscle cells using fluorescent dyes.  
: >
: >Does anyone have experience with this type of measurement?
: >Can you give any hints as to the application of the dyes,
: >specificity of measurement, sensitivity, suitability of
: >the dyes?
: >

: I don't have any experience with this type of experiments,
: but I think the only way do do this so far is by using
: recombinant aequorin as described by Rizzuto, R., Simpson, A.W.M., 
: Brini, M., and Pozzan, T. Rapid changes of mitochondrial Ca2+ 
: revealed by specifically targeted recombinant aequorin. 
: Nature 358:325-327, 1992. 

	You can use fluorescent dyes (fura-2 or indo-1)
See Miyata et al. (1991) Measurement of mitochondrial free Ca2+
in rat cardiac myocytes  Am. J. Physiol. H1123-34.

The technique relies on the loading of the membrane permeant form
of the dye into non-cytosolic compartments such as the mitochondrial.
Following, the fluorescence of the cytosolic dye is quenched by
Mn2+ (100 micromolar), and the residual fluorescence should reflect
mitochondrial fura-2/indo-1 fluorescence (assuming that deesterification
of the dye is complete).  The technique has been used with both isolated
cardiac myocytes and isolated hearts.  I have used it myself to 
determine the portion of the dye in the mitochondria.

	Potential problems:
	1. entry of the dye into the mitochondria
	2. alteration of excitation-contraction coupling by Mn2+
	3. mitochondrial [Ca2+] outside the sensitivity range of the
	   fura-2 or indo-1, but Miyata et al., I believe, report
	   a measurement of 500nM, which is close to the Kd of fura-2
	   or indo-1.
	4. contribution of the sarcoplasmic reticulum.  However, the
	   SR is roughly 3% of the cell volume, suggesting no
	   major contribution.

	I note that you state calcium content rather than [Ca2+] in which
case, I don't know, though electron probe microanalysis been used to estimate
mitochondrial calcium content.  (see Isenberg et al. (1993) Measurement of
changes in mitochondrial calcium concentration during the cardiac calcium
cycle Cardiovascular Research 27:1800-9)

	Good luck

Richard Kondo
--
kondo at buphy.bu.edu
Cardiac Muscle Research Laboratory
Boston University




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