Dear Don,

Thanks Don. I certainly agree with your point that dyes may have
different Kd's and/or spectral characteristics in different environments
(eg. cytosol and nucleus). This is a possibility in explaining
observations of apparent nuclear [Ca++] elevations. I applaud nearly
every thing you say. I still have trouble accepting the term "ALWAYS"
(your emphasis) in your original posting.

>While I haven't looked at nuclear calcium signals in
>T cells, to my knowledge, nuclear pore structures are well
>conserved across vertebrate cell types and the pore is ALWAYS
>freely permeable to calcium.

The nuclear-cytosolic Ca++ gradient was totally abolished with an
ionophore in the Williams et al (1985) paper. Suggesting that the
gradient across the nuclear envelope in this study was probably real.
We've also observed similar results with the pH indicator SNARF-1 where
the apparent difference between the nucleus and cytosolic pH can be
totally abolished with ionophores Dubbin et al. (1993). Mind you in
these experiments, in a few individual cells, while the pH gradient was
reduced substantially, it was not totally abolished. Indicating that the
dye may have been "behaving" differently in some of the nuclei. Thus
supporting some of your arguments.

One of the things that struck me about Nuclear Ca++ waves in cardiac
cells was that the elevation of calcium in the nucleus consistently
followed that of the cytoplasmic calcium fairly closely. However, the
uptake time varied considerably from cell to cell. One hypothesis we
proposed was that Ca++ may be release directly into the nucleus from the
nuclear envelope (this being continuous with the sarcoplasmic
reticulum). The variation in the return to resting nuclear calcium
levels might be explained by the state of the nuclear pores and their
resistance to calcium. If the nuclear pores are open calcium should rise
and fall quickly with the cytosol. If the pores are 'closed', and
calcium may be released directly into the nucleus from the nuclear
envelope. Then the rise in calcium will be almost immediate, but the
return to resting levels may take considerably longer.

I've enjoyed this discussion but apologise to those who have no interest
in this field.

Cheers
Stephen Cody

Williams, et al. (1985) Calcium gradients in single smooth muscle cells
revealed by the digital imaging microscope using Fura-2. Nature, 318:
558-561.

Dubbin et al. (1993). Intracellular pH mapping with SNARF-1 and confocal
microscopy.  II: pH gradients within single cultured cells. Micron,
24:581-586.


*************** New Address ***************
Stephen H. Cody,
Colon Molecular and Cell Biology Laboratory,
Ludwig Institute for Cancer Research,
Post Office Royal Melbourne Hospital,
Parkville, Victoria 3055, Australia.