[See ref 21
]
If mitochondria are the best candidates for the natural emitters of light pulses,
they should express some of the fundamental properties of other known generators
of pulse trains, including the existence of 2 levels of light emission, a threshold
level of excitation that causes them to leap from a low level of emission to a high
level, and a refractory period during which they return to the ground state. Studying
the autofluorescence properties of mitochondria which were excited with 365 nm UV
light we found, indeed, all these properties expressed in mitochondria. For these
experiments we used the following self-built micro-spectrograph.
Basic design of the micro-spectrograph(1): light source (50 W mercury lamp),
(2): excitation filter (365 nm), (3):condensor, (4): stage and specimen,
(5): 25x objective lens, (6): mirror, (7): slit (< 1mm), (8): barrier filter (transmits >400 nm),
(9): lens to create image of slit (= spectrum), (10): 60o glass prism to generate spectrum,
(11), (13): 1:1 imaging lenses of baffle, (12): N2 flushed baffle,
(14): liquid N2 cooled CCD camera, (15): typical fluorescence spectrum of live cells seen by the CCD camera.
.
The excitation-induced enhancement of fluorescence
During continuous irradiation for 2-3 minutes with ultraviolet light ( 365 nm)
one or several peaks of the autofluorescence spectrum flared up 3-fold or
more. Subsequently the peaks remained stable at this new level of intensity for 30 min or longer. Within the limits of resolution of ±10 nm of the micro-spectrograph
the location of the emission peaks did not change.There are numerous wavelengths capable of stimulating
the autofluorescence of mitochondria. These wavelengths appear to be related to the absorption spectra
of flavins and NADH. The above observation is remarkable, because it points to an additional mechanism
that enhances the mitochondrial fluorescence with increasing time.
Fluorescence enhancement of 3T3 cells at 37o C on a cover slip irradiated
with near-ultraviolet light ( l = 365±10 nm; 16 mW/mm2 ). The numbers at the top of each panel
indicate the duration of irradiation in minutes. The 3 panels on the right are the spectra of narrow
band interference filters (peaks of transmission indicated on the panels) recorded under identical
conditions as the cells.
(Animated version of the above experiment)
Reversibility of level transition
The effect was reversible, i.e. (a) the induced increase of fluorescence could be reversed by turning
off the excitation light for a certain period of time, and (b) the effect could be re-stimulated using
the same cells. Therefore, the effect was not due to UV damage of the cells. As shown in detail in the
full article, it appears that the enhancement of autofluorescence is due to an increase of the quantum
efficiency of the fluorescence mechanism. Based on these results, one may characterize the effect as
'Reversible, Excitation Light-Induced Enhancement of Fluorescence'. In order to simplify the text below,
we shall use the acronym 'RELIEF'.
Recovery of CV1 cells from 18 min long RELIEF (= 3x the necessary duration of
UV irradiation). After turning off the excitation for a recovery period of 30 min, RELIEF could be
stimulated again using the identical cells.
Evidence for mitochondrial origin of RELIEF
RELIEF was found to be insensitve to drugs that inhibit protein synthesis or cytoskeletal elements.
In contrast, it was inhibited by drugs that inhibited mitochondrial function. Since the short exposures
of cells to the mitochondrial inhibitors were not sufficient deprive the cells of ATP,
the results suggest that RELIEF may be an expression of intact mitochondria, and not merely an
energy dependent phenomenon.Consistent with this interpretation that RELIEF originated in the mitochondria,
cells or cell fractions without mitochondria not express RELIEF. On the other hand, destructive cell
treatments that left mitochondria or at least their inner membrane intact continued to support RELIEF.
For example, cell homogenates generated by intense douncing of rat liver expressed RELIEF very intensely.
Preliminary studies showed that preparations of mitochondria from mouse liver did not express RELIEF.
Instead, the fluorescence faded. However after suspending the mitochondria in normal culture medium to
which 10 mM Na-pyruvate was added, the fluorescence recovered within 4 minutes and, subsequently, expressed RELIEF.
Other systems expressing RELIEF
In addition to 3T3 and CV1 cells we found that cells from other placental mammals such as HeLa cells
and BHK cells expressed RELIEF, as well. Not only cultured cells, but also cells isolated directly from
tissue expressed RELIEF. For example, rat liver homogenates expressed RELIEF very strongly.
When we tested cells from organisms other than placental mammals, we did not find expression of RELIEF.
The tested celltypes included avian cells (chick embryo fibroblasts SL-29), amphibian cells (homogenates
of Xenopus laevis liver), insect cells ( Drosophila melanogaster cell line Kc167), nematode cells (intact
worms and homogenates of Caenorhabditis elegans), yeast cells (Saccharomyces cerevisiae), and slime mold
cells (Dictyostelium discoideum).In addition, PtK1 cells (Potorous tridactylis) were tested as an example
of marsupial mammalian cells.
If the mitochondria are the sources of RELIEF, one may expect that all eukaryotic cells express it.
In contrast, the above results suggested that only placental mammalian cells contained mitochondria that were
capable of expressing RELIEF under the described experimental conditions. The mitochondria of other cells
may not express it, or else they may require other excitation conditions.
Conclusion
The 365 nm photons that triggered the unknown function underlying RELIEF are hardly its natural trigger.
It seems more likely, that they reflect the height of an energy barrier that was overcame 'by force' in
the above experiments, whereas the cells under normal conditions can be expected to bypass it by a
specific enzymatic reaction whenever they trigger the unknown function.
In order to aid the future search for the unknown function underlying RELIEF, one may try to design
its profile as follows. It appears to
a. mediate the transition between a resting and an active state (bi-stability),
b. be protected by a threshold, and
c. recover from the transition with a certain refractory period (refractory behavior).
Nerve excitation and other pulsating phenomena shares the above 3 properties with the unknown function,
albeit on a very different time scale. Therefore, the results may point to the existence of an excitation
mechanism for (mammalian?) mitochondria which, in turn, may be involved in the generation of pulsating (light?)
signals of these mitochondria and, thus, of the corresponding cell types. This hypothesis is illustrated below.
Illustration of the light-pulse generator hypothesis of mitochondria. The resting state
of the mitochondria is elevated to the active state by either a 365 nm photon in the experimental
situation described here, or by an unspecified enzyme action in the in vivo situation of the cell.
After emitting light (600nm in the experimental situation described here or 800 nm in the in vivo
situation of the cell) the mitochondria return to the resting state during a refractory period.
[See ref 21].