Dynamic Changes in Spontaneous Intracellular Free Calcium Oscillations and Their Relationship to Prolactin Gene Expression in Single, Primary Mammotropes
Carlos Villalobos,
William J. Faught and
L. Stephen Frawley
Laboratory of Molecular Dynamics Department of Cell Biology and
Anatomy Medical University of South Carolina Charleston, South
Carolina 29425
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ABSTRACT
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Cytosolic calcium plays a critical role in the
control of a number of genes, including that of the pituitary hormone
PRL. Cells that secrete this hormone, termed mammotropes, display
spontaneous oscillations of intracellular free calcium
([Ca2+]i) that are
positively correlated to PRL release. However, the precise contribution
of calcium signaling to the expression of any gene including PRL has
remained obscure owing to the requirement for and lack of a strategy
for monitoring both of these dynamic variables (gene expression and
[Ca2+]i oscillations)
in the same living cell. In the present study, we overcame this
technical limitation by making real-time measurements of PRL gene
expression in transfected, primary rat mammotropes previously subjected
to [Ca2+]i
determinations by digital imaging fluorescence microscopy of fura-2.
Our results showed that the majority of mammotropes (75%) exhibited
distinct oscillatory behaviors that could be subgrouped on the basis of
frequency/amplitude of
[Ca2+]i changes,
whereas the remainder (25%) were quiescent (nonoscillatory).
Interestingly, most mammotropes displayed spontaneous transitions
between oscillatory and quiescent states over the course of several
hours. As a consequence of this oscillatory plasticity, there was not a
positive correlation between
[Ca2+]i dynamics and
gene expression at any point in time, as would be predicted by studies
with entire populations of cells. Instead, the relationship was
distinctly inverse, suggesting that dynamic changes in PRL gene
expression may be regulated by temporally dissociated transitions
between quiescent and oscillatory states.
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INTRODUCTION
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Dynamic changes in the concentration of intracellular free calcium
([Ca2+]i) play a pivotal role in the control
of many physiological functions (1). One of these is stimulus-secretion
coupling, and the PRL-secreting mammotrope has been studied extensively
in this regard (2, 3, 4). Indeed, mammotropes, like other pituitary cells,
display spontaneous oscillations of [Ca2+]i
that are driven by electrical activity and modulated by a variety of
hypophysiotropic agents (5). Such oscillations are positively
correlated with hormone release, in that exocytotic events are
invariably associated with increased oscillatory activity (6). There is
also ample evidence to indicate that cytosolic calcium imposes a major
regulatory influence on the initiation of the PRL biosynthetic
processes as well as the final step. The case for calcium regulation of
transcription in mammotropes is compelling and derives from
observations that physiological agonists (e.g. TRH) that
stimulate PRL gene expression also evoke increases in
[Ca2+]i in pituitary cell lines and normal
mammotropes (7), and conversely, that abolition of spontaneous or
induced [Ca2+]i oscillations (by removal of
extracellular calcium or exposure to calcium antagonists) decreases PRL
gene expression (8). Moreover, the PRL gene promoter possesses several
regulatory sequences that are reported to confer transcriptional
responsiveness to calcium (9, 10). Thus, calcium functions as a
critical second messenger that controls key regulatory checkpoints that
span the entire length of the PRL secretory pathway.
Our depth of knowledge about the regulatory role of calcium in
stimulus-secretion coupling is attributable largely to the fact that
many investigations have been carried out at the single cell level to
obviate the confounding effects of heterogeneous responses. Thus, the
capacity to make combined measurements of exocytosis (by changes in
membrane capacitance or reverse hemolytic plaque development) and
[Ca2+]i oscillations (by microfluorimetry or
electrophysiological measurements) on the same cell has contributed
greatly to our knowledge about the temporal aspects of calcium dynamics
and hormone export (11, 12, 13). Unfortunately, our level of understanding
about the relationship between [Ca2+]i
changes and PRL gene expression is less well developed due to the fact
that the consensus experimental paradigm in this area has been to
pharmacologically manipulate [Ca2+]i within
entire cultures of pituitary cells and then measure steady state levels
of PRL messenger RNA (mRNA) by Northern analysis (14, 15). Although
much has been learned with this approach, further progress would be
facilitated greatly by access to a single cell strategy that would
enable the monitoring of these two extremely dynamic variables
([Ca2+]i and gene expression) in the same
cell. In the present study we circumvented this technical constraint by
subjecting the same individual, living mammotropes to digital imaging
fluorescence microscopy of fura-2 (for monitoring
[Ca2+]i dynamics) followed by real-time
measurement of PRL gene expression (16). The latter technique, which is
based on quantification of photonic emissions emanating from single
mammotropes transfected with a PRL promoter-driven luciferase
construct, provides a reliable estimate of the relative level of gene
activity within a living cell (17). Results obtained with this combined
approach show, interestingly, that calcium oscillatory behavior within
a mammotrope can vary greatly over time. As a consequence, there is
not a positive correlation between oscillatory activity and PRL gene
expression in single mammotropes at a given point in time, as would be
predicted by studies with entire populations of cells.
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RESULTS
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Characterization of Spontaneous
[Ca2+]i Oscillations in Normal
Mammotropes
To establish oscillatory subtypes, we subjected anterior pituitary
(AP) cells in primary culture to [Ca2+]i
measurements by digital imaging fluorescence microscopy for about 30
min. Mammotropes were identified post-facto by their ability
to express the luciferase structural gene driven by the PRL promoter.
We found that all identified mammotropes exhibited one of the four
patterns illustrated in Fig. 1
. To be
specific, about a quarter of the cells showed no spontaneous
[Ca2+]i oscillations during the entire
[Ca2+]i measurement period (quiescent cells;
Fig. 1A
). The remaining cells exhibited discrete patterns of
spontaneous [Ca2+]i oscillations, the most
common of which was characterized by high frequency, low amplitude
[Ca2+]i transients (Fig. 1B
). A less common
pattern was characterized by high frequency, high amplitude
[Ca2+]i oscillations (Fig. 1C
). Finally, a
minority subset of mammotropes displayed slow waves of
[Ca2+]i (Fig. 1D
). For quantification of
[Ca2+]i oscillations in normal mammotropes,
we employed two parameters previously developed to analyze
[Ca2+]i levels and oscillations in pituitary
cells: the mean [Ca2+]i value and the
oscillation index (18, 19). The former represents an average of the
[Ca2+]i values during the measurement period.
The oscillation index reflects an average of the changes in
[Ca2+]i and is largely independent of the
actual level of [Ca2+]i. Table 1
shows the averaged values of mean
[Ca2+]i and oscillation index for the cells
pooled according to the various [Ca2+]i
oscillatory patterns shown in Fig. 1
.

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Figure 1. Patterns of Spontaneous
[Ca2+]i Oscillations in Normal
Mammotropes
AP cells in primary culture were subjected to
[Ca2+]i measurements for 30 min. Mammotropes
were identified thereafter by their ability to express the luciferase
structural gene under the control of the PRL gene promoter, as stated
in Materials and Methods. Cells were categorized into
subgroups on the basis of whether they were quiescent (nonoscillating;
A), or exhibited one of three oscillatory phenotypes (BD). Cells
displaying [Ca2+]i changes higher than 50
nM but lower than 500 nM were assigned to the
low amplitude, high frequency group (B). For the high amplitude, high
frequency category, we selected those cells exhibiting
[Ca2+]i changes higher than 500
nM (C). Finally, those cells exhibiting slow waves of low
amplitude [Ca2+]i oscillations comprised
their own category (D). The examples shown are representative of those
obtained when 112 mammotropes were studied in 10 independent
experiments.
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Relationship between [Ca2+]i
Oscillations and PRL Gene Expression in Individual Mammotropes
Having established the heterogeneous nature of
[Ca2+]i oscillations in normal mammotropes,
we next attempted to elucidate the relationship between
[Ca2+]i oscillations and transcriptional
activity of the PRL gene in the same living mammotropes. To this end,
AP cells previously microinjected with the rat PRL-LUC plasmid and used
for [Ca2+]i measurements were subjected
immediately thereafter to photon counting for quantification of PRL
gene expression. The results of this combined protocol are illustrated
in Fig. 2
. Specifically, a fluorescence
image was captured after [Ca2+]i measurements
to identify the cells studied (Fig. 2A
). Then, specific photonic
emissions from the same cells were quantified, and the resulting data
were stored as computer files for latter analysis (Fig. 2B
). To explore
a possible relationship between [Ca2+]i or
[Ca2+]i oscillations and PRL gene expression,
specific photonic emissions for each cell were plotted against its
corresponding mean [Ca2+]i value (Fig. 2C
) or
oscillation index (Fig. 2D
). Consistent with our previous observation
(17), we found that basal expression of the PRL promoter-driven
reporter construct varied considerably from cell to cell, with values
ranging from 5010,000 specific photonic emissions/10 min·cell.
Interestingly, cellular values for mean
[Ca2+]i and oscillation index were likewise
extremely heterogeneous, in that they ranged from 2 to about 100
nM/s for both parameters. Surprisingly, there was not a
positive correlation between the degree of PRL gene expression
(photonic emissions) and either of the two calcium parameters
quantified. However, it is noteworthy that the highest levels of PRL
gene expression were generally associated with cells exhibiting the
lowest values of mean [Ca2+]i or oscillation
index. A possible relationship between oscillatory phenotype and gene
expression was next explored by calculating the average level of
photonic emissions for each of the oscillatory subtypes described in
Fig. 1
. As shown in Table 1
(last column), the relative
degree of gene expression was very similar for the three categories of
mammotropes that exhibited spontaneous
[Ca2+]i oscillations (groups B-D). However,
when these values were considered collectively and compared with those
of their nonoscillatory counterparts, the latter was found to be 2-fold
higher than that of the former (Fig. 3
).
In contrast, mean [Ca2+]i values were about
3-fold greater for oscillatory than for nonoscillatory cells (Fig. 3
).
Thus, at any point in time, there was a distinct inverse relationship
between [Ca2+]i and PRL gene expression
within individual cells.

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Figure 2. Concurrent Measurements of
[Ca2+]i and PRL Gene Expression in the Same
Cells
AP cells within a grid were microinjected with a plasmid containing the
luciferase structural gene under the control of the PRL gene promoter.
After 2 days, microinjected cells were relocated and subjected to
[Ca2+]i measurements by digital imaging
fluorescent microscopy for 30 min. Then, a fluorescence image was
captured for reference purposes (A). Immediately after
[Ca2+]i measurements, the same cells were
subjected to quantification of luciferase/luciferin-generated photonic
emissions reflective of PRL gene expression (B). These photonic
emissions were plotted against the mean
[Ca2+]i level (C) or oscillation index (D)
computed from the [Ca2+]i traces obtained
from the same cells. The data shown are from 10 independent experiments
(n = 112 cells).
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Figure 3. The Level of PRL Gene Expression Is Higher in
Quiescent Mammotropes Than in Cells Exhibiting Spontaneous
[Ca2+]i Oscillations
All of the mammotropes studied were pooled into two groups: quiescent
(n = 28) and oscillating cells (n = 84). Specific photonic
emissions reflective of PRL gene expression (solid bars)
and mean [Ca2+]i values (open
bars) were then averaged for both groups. Quiescent cells
exhibited a significantly higher level of PRL gene expression than
those cells displaying oscillations of
[Ca2+]i (*, P < 0.05
vs. oscillating cells).
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One might argue that the observed relationship between PRL gene
expression and calcium dynamics is attributable to the protracted
half-life of luciferase in our system and/or the time required for
synthesis of new luciferase reporter enzyme. The former possibility can
be discounted by the data presented in Fig. 4
, which shows that the half-life of
luciferase in primary mammotropes is relatively short (
60 min). The
issue of the time course for luciferase synthesis is extremely
difficult to address directly because we currently lack of a method
capable of discriminating recently synthesized luciferase from that
already present in the same living cell. Nevertheless, one way to
approach the problem is to measure the time required to first detect
light after transfection of cells with the PRL promoter-driven
luciferase plasmid, and our results indicate that this can frequently
be achieved within the first hour after transfection (data not shown).
Thus, the time required for synthesis and degradation of reporter
enzyme might contribute to the lack of correlation between gene
expression and calcium dynamics, but probably not to a major
extent.

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Figure 4. Functional Half-Life of Luciferase
AP cells in primary culture were microinjected with purified firefly
luciferase protein (Sigma) at a dose (1 mg/ml), which was designed to
achieve a level of photonic activity comparable to that detected in
cells transfected with our reporter plasmid. Immediately after
microinjection, coverslips were immersed in luciferin-containing medium
and placed in our photon-counting system to monitor luciferase activity
over time. The mean activity of 30 single cells is shown. The results
obtained in this experiment are representative of those from two
others.
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Spontaneous [Ca2+]i
Oscillations Can Fluctuate over Time
One possible explanation for the lack of positive correlation
between [Ca2+]i oscillatory behavior and PRL
gene expression is that the former may change over time. In this
scenario, the level of gene expression at any particular point in time
might actually reflect changes in [Ca2+]i
oscillatory behavior that occurred up to several hours earlier. To test
this line of reasoning, we subjected the same identified mammotropes to
[Ca2+]i measurements on three separate
occasions over a 24-h period. Representative examples of the results
obtained are presented in Fig. 5
. We
found that most cells maintained the same
[Ca2+]i oscillatory pattern over a 3-h period
(e.g. Fig. 5
, cells A, C, and D), although some mammotropes
changed their [Ca2+]i oscillatory pattern
during this time frame (e.g. cell B in Fig. 5
). In contrast,
cells exhibited dramatic changes in oscillatory patterns over longer
(24-h) periods of time (see cells A, B, and C as opposed to D in Fig. 5
). To be more specific, the fraction of cells that changed from one of
the four oscillatory patterns to another increased from 22% between
03 h to 60% between 024 h (Fig. 6
).
Further analysis of the same dataset revealed that the majority of
these changes (50% and 75% at 3 and 24 h, respectively) was
attributable to transitions between quiescent and oscillatory states or
vice versa, rather than to changes among the four basic
oscillatory phenotypes. Despite the oscillatory plasticity exhibited by
individual mammotropes, the relative proportion of all cells that
displayed the four [Ca2+]i oscillatory
patterns (illustrated in Fig. 1
) remained remarkably constant during
the three [Ca2+]i measurement periods (data
not shown). These results indicate clearly that spontaneous
[Ca2+]i oscillations remain quite constant
over a few hours, but tend to change over longer periods. Moreover,
most transitions result from excursions between quiescent and
oscillatory states.

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Figure 5. Individual Mammotropes Exhibit Changes in the
Pattern of [Ca2+]i Oscillations over Time
Identified mammotropes were subjected to 10-min
[Ca2+]i measurements at the three times
indicated. The pattern of [Ca2+]i
oscillations changed over time in most mammotropes. These four traces
(AD) are representative examples of 56 individual mammotropes that
were studied in three independent experiments.
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Figure 6. Percentage of Mammotropes That Exhibit Changes in
the Patterns of [Ca2+]i Oscillations over
Time
Mammotropes that were subjected to [Ca2+]i
measurements at three separate times (0, 3, or 24 h) were analyzed
to determine whether they changed their
[Ca2+]i oscillatory pattern from one of the
four identified previously (Fig. 1 ) to another. After 3 h, only
22% of the mammotropes changed their pattern of
[Ca2+]i oscillations. This percentage
increased to 60% after 24 h. The percentage of cells exhibiting
transitions between quiescent and oscillatory patterns represented 50%
and 75% of all of the changes at 3 and 24 h, respectively.
Therefore, transitions between quiescent and oscillatory patterns
account for the majority of changes observed.
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DISCUSSION
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The overall goal of this study was to elucidate the relationship
between calcium dynamics and PRL gene expression in single mammotropes.
A priori, we envisioned at least three aspects of calcium
signaling that might be relevant to regulation of the PRL gene: the
overall pattern of [Ca2+]i oscillations, the
mean [Ca2+]i value, and the oscillation
index. Inasmuch as all three variables can be quantified from the same
dataset, we chose as a point of departure an experiment aimed at
characterizing in a narrow frame time (30 min) the overall patterns of
[Ca2+]i oscillations in single living
mammotropes. We found that the majority (75%) of mammotropes from
lactating rats exhibited distinct oscillatory behaviors that were
amenable to subgrouping on the basis of differences in the
frequency/amplitude of [Ca2+]i changes. In
contrast, a minority subset accounting for only 25% of all the
mammotropes studied were relatively quiescent (i.e.
nonoscillatory). Our results differ from those of Hinkle and co-workers
(20), who found that spontaneous oscillations were common among clonal,
PRL-secreting GH3 cells (63%), but were rarely observed in
primary cultures derived from retired breeder, female rats (21). Lewis
et al. (22), on the other hand, found that 22% of cycling
female mammotropes displayed oscillations, and Ho et al.
(23) reported a value of 43% for mammotropes derived from the same
physiological model. Given that oscillatory activity is positively
correlated to secretion (24), and that the predicted rank order for the
rate of PRL export from mammotropes is lactators > cycling
females > retired breeders, it seems reasonable to propose that
the preponderance of spontaneous oscillators observed in the present
study is a simple reflection of the highly active secretory status of
mammotropes from lactators.
The time-resolved analysis of calcium dynamics and gene expression in
the same cell was made possible by the combined application of digital
imaging fluorescence microscopy of fura-2 with real-time measurement of
PRL gene expression. That the latter strategy provides a reliable
estimate of gene activity in living cells is evidenced by our previous
observations that expression of the transfected reporter construct is
highly cell specific (restricted to mammotropes in this instance) and
that treatments with agents known to either increase or decrease PRL
gene expression by entire populations of cells have identical,
predictable effects on the rates of photonic emissions from single,
transfected mammotropes (17). Moreover, our observation that the
functional half-life of luciferase in this system is fairly short (60
min) lends confidence that rapid changes in the rate of gene expression
(reflected by newly synthesized luciferase) would not be obscured by a
high background of preexisting reporter activity. Armed in the present
study with this powerful and responsive tool, we measured PRL gene
expression in living cells previously subjected to measurement of
calcium dynamics and found, quite surprisingly, that these two
variables were not positively correlated. In fact, the relationship was
distinctly inverse regardless of the measure of internal calcium
activity used for comparison with the relative index of gene expression
(photonic emissions). These results cannot be interpreted to mean that
calcium is unimportant for the regulation of PRL gene expression, for
if that were the case, one would expect to find no correlation between
these two variables as opposed to a negative one. The distinct inverse
correlation between these variables measured in the same living cell
might indicate that a given mammotrope temporally dissociates hormonal
secretion (which is associated with an increased frequency of calcium
oscillations) from biosynthesis. In this scenario, a particular series
of [Ca2+]i oscillations would act over the
short term to evoke hormone release and over the long term to induce
gene expression required for replenishment of hormonal stores. Our
recent observation on the dissociation between PRL gene transcription,
mRNA storage, and hormone release within the same mammotropes is
entirely consistent with this possibility (25).
Our present findings on single, primary mammotropes are difficult to
reconcile with those from population studies (largely with tumor cell
lines) that suggest that calcium is a pivotal modulator of PRL gene
activity. As indicated above, one possible explanation for these
seemingly disparate observations is that a given mammotrope might
change its [Ca2+]i oscillatory pattern from
time to time, and that changes in PRL gene expression induced by such
signals would naturally lag behind, owing to the fact that oscillatory
phenotypes could conceivably change in seconds, whereas modulation of
gene expression would require many minutes or even hours for full
manifestation. When we tested this idea experimentally, we found that
individual mammotropes could indeed change their oscillatory phenotype
over the course of just a few hours, although they were more likely to
do so from one day to the next. Most interestingly, the vast majority
of such transitions occurred between quiescent and oscillatory
phenotypes as opposed to between one oscillatory subtype and another.
To our knowledge, this is the first report that normal mammotropes in
primary culture have the capacity to switch spontaneously between
quiescent and oscillatory states. Whether this also occurs in
vivo remains to be established. Parenthetically, it is noteworthy
that individual mammotropes from lactating rats undergo striking day to
day fluctuations in both the relative amount of PRL secreted (26) and
the basal level of PRL gene expression (17). On the basis of these
collective observations, it is tempting to speculate that spontaneous
transitions in the calcium oscillatory behavior of mammotropes may
mechanistically under- lie day to day variations that occur in both
of these calcium-dependent phenomena.
How might varying the mode of calcium presentation be advantageous to
the physiological regulation of PRL gene expression in living
mammotropes? This question is impossible to answer directly because so
little is known about the relationship between calcium dynamics and PRL
gene expression in single cells, particularly those from normal
pituitary glands. Nevertheless, a series of cleverly designed
population studies conducted by Haisenleder and co-workers (27) has
provided important insights about how these processes might interact in
the same untransformed cell. Briefly, these investigators perifused
pituitary cells (from adult female rats) with various agents known to
modulate the pattern of calcium influx (KCl, the calcium channel
activator BayK 8644, or the calcium ionophore A23187). These treatments
were administered for 24 h in either a pulsatile or a continuous
manner, after which the perifused cells were recovered, extracted, and
subjected to PRL mRNA determinations. Interestingly, these
investigators found that the agents could either increase or decrease
steady state levels of PRL mRNA depending on the mode of presentation
(pulsatile or continuous). To be more specific, they observed that
intermittent elevation of [Ca2+]i was much
more effective at augmenting PRL gene expression than persistent
induction of calcium influx across the mammotrope membrane, which had a
profound inhibitory effect (27). These data clearly reinforce the idea
that a pulsatile calcium signal is required to stimulate PRL gene
expression in mammotropes. By extension, it seems reasonable to propose
that spontaneous transitions between oscillatory and nonoscillatory
states comprise the mechanism by which a mammotrope can optimize
calcium stimulation of PRL gene expression while avoiding the
deleterious effects of continuous calcium exposure.
The existence of [Ca2+]i oscillations that
vary in frequency, amplitude, and/or shape has led to the proposal that
calcium-dependent processes are regulated by qualitative and/or
quantitative modulation of oscillatory
[Ca2+]i signal characteristics (28, 29, 30). In
this scenario, extracellular signals would be transduced to a specific
calcium code that subsequently would be deciphered by a sensor
associated with the effector system (31). Our present observations on
the phenotypic heterogeneity of calcium oscillations in PRL cells, the
temporal dissociation between calcium oscillatory activity and PRL gene
activity, and the dramatic effect that frequency modulation of
[Ca2+]i has on PRL gene expression (28) all
provide compelling (albeit indirect) evidence that such a coding system
is operative in normal mammotropes. Unfortunately, the precise nature
of this putative code has defied resolution because the relevant
variables (patterns of gene expression and calcium oscillations) are
essentially "moving targets." It is becoming abundantly obvious
that success in cracking the calcium code will require the monitoring
of both dynamic variables multiple times (if not continuously) in the
same living cell. Although technically onerous, this is currently our
approach for ongoing investigations in this research area.
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MATERIALS AND METHODS
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Cell Dispersion and Microinjection
Monodispersed AP cells from primiparous, lactating (days 610
postpartum) rats (Sprague-Dawley Harlan, Madison, WI) were prepared as
described previously (17). Cells were plated onto
poly-L-lysine-coated, gridded coverslips at a density of
75,000 cells/75 µl of a defined medium [phenol red-free medium
199/nutrient mixture F-12 (1:1; Life Technologies, Grand Island, NY) in
which L-valine was replaced by D-valine]
supplemented with 0.1% BSA, insulin-transferrin-selenium premix, and
antibiotics. Cells were incubated for 1 h to facilitate
attachment, covered with 2 ml defined medium supplemented with 10%
FBS, and cultured in a humidified atmosphere of 5%
CO2-95% air for 2 days. The cells within a particular grid
were then microinjected with a reporter plasmid (0.2 µg/µl in 10
mM PBS) in which 2.5 kilobase pairs of the 5'-flanking
region of the rat PRL gene were placed upstream of the coding sequence
for firefly luciferase. After microinjection, cells were washed twice
and cultured for 2 more days in phenol red-free DMEM (Life
Technologies) supplemented with 10 mM HEPES, 10% FBS,
0.1% BSA, and antibiotics.
[Ca2+]i Measurements in
Single, Identified Mammotropes
[Ca2+]i measurements were performed on
primary cultures of AP cells loaded with the calcium-sensitive probe
fura-2 (32). This was accomplished by digital imaging fluorescence
microscopy essentially as reported previously (33). Briefly, cells were
loaded with 2 µM fura-2/AM (Molecular Probes, Eugene, OR)
for 1 h at 37 C in culture medium (Sigma Chemical Co., St. Louis,
MO) of the same composition as before except that it was devoid of FBS,
BSA, and bicarbonate. Coverslips were then washed three times in the
same medium lacking fura-2/AM and mounted over the heated stage (37 C)
of an Axiovert 35 inverted microscope (Zeiss, Jena, Germany). The
microscopic field containing microinjected cells was then reidentified
(from its position on the numbered/lettered, gridded coverslip), and
the cells were epiilluminated alternately at 340 and 380 nm. Emission
of light above 520 nm was recorded and analyzed with an Attofluor Ratio
Vision System (Atto Instruments, Rockville, MD). Two video frames of
each wavelength were averaged with an overall resolution time of 4 sec
for each pair of images at alternate wavelengths. The ratio of
consecutive frames obtained at 340 and 380 nm excitation light was
calculated, and [Ca2+]i was estimated by
comparison with fura-2 standards (32).
Measurements of PRL Gene Expression in Single, Living
Mammotropes
Within 10 min of completing [Ca2+]i
measurements, the coverslip containing AP cells was assembled into a
Sykes-Moore chamber and transferred to the heated stage (37 C) of a
photon capture system (17) for quantification of
luciferase-luciferin-generated photonic emissions. This system was
comprised of a Zeiss Axioskop in series with a Hamamatsu VIM photonic
camera/Argus 50 image processor. To be more specific, the microscopic
field in which [Ca2+]i measurements were just
performed was reidentified, and a brightfield image was captured for
reference purposes. Next, 3 ml of the same medium used for
[Ca2+]i measurements were supplemented with 3
mM luciferin along with 1% dimethylsulfoxide and infused
into the chamber (total volume, 0.75 ml). Seventeen minutes later,
photonic signals emitted by individual cells were accumulated over a
10-min period, and the images obtained were stored as computer files.
For quantification of photonic events, the image of accumulated
photonic emissions was superimposed over the brightfield image of
individual cells, and the number of photonic events within a window of
fixed area was calculated. Photonic measurements made in at least 20
adjacent areas devoid of cells were used to compute a background value.
This was subsequently subtracted from the total accumulation to
calculate specific photonic emissions from each cell.
It is noteworthy that expression of the transfected reporter plasmid
used in this study is specific to mammotropes. Indeed, we had shown
previously (17) that all transfected pituitary cells that emitted
photons after exposure to luciferin contained PRL
(immunocytochemistry), released the hormone (reverse hemolytic plaque
assay), or contained the corresponding mRNA (in situ
hybridization cytochemistry). It should also be mentioned that the
presence of fura-2 did not interfere with our bioluminescence
measurements, as the latter were performed in complete darkness (no UV
excitation), and the background values of photonic emissions did not
differ for fura-2-loaded and nonloaded cells (data not shown).
Multiple Measurements of
[Ca2+]i from the Same Cell
In those experiments in which [Ca2+]i
dynamics were monitored on three separate occasions from the same
transfected cells, coverslips were washed three times in culture medium
after the first [Ca2+]i measurement and
placed in the incubator for 2 h while immersed in the same medium
supplemented with 0.1% BSA and 10% FBS. Next, cells were loaded again
with fura-2/AM as described above and subjected to a second period of
[Ca2+]i measurements. The coverslips were
then subjected to photonic imaging to identify which of the cells under
study were mammotropes (as determined by their ability to emit light
after exposure to luciferin). Finally, the cells were washed, incubated
for 23 h in the same culture medium, and subjected again to a
period of loading with fura-2/AM, followed by
[Ca2+]i measurements. In all instances,
[Ca2+]i monitoring was restricted to a 10- to
15-min period to optimize the viability of cells. Relocation of the
same cells was achieved by the combined use of the photoengraved
coverslips and computer retrieval of information about relative
positions. Cells that were not positively identified as mammotropes or
that had changed their location were excluded from the analysis.
Quantification of [Ca2+]i
and [Ca2+]i Oscillations
Two relevant parameters were calculated for quantification of
[Ca2+]i levels and
[Ca2+]i oscillations in identified
mammotropes as reported previously (18, 19): the mean
[Ca2+]i value and the oscillation index. To
calculate the mean [Ca2+]i value,
measurements of [Ca2+]i obtained at 4-sec
intervals were accumulated and normalized for the length of the entire
sampling period. Thus, this parameter provides a mean value of
[Ca2+]i over time. The oscillation index
represents the rate of changes in [Ca2+]i
during the period of measurement. To determine this, absolute
differences in [Ca2+]i levels between
successive measurement intervals were averaged for the time period
during which data were collected. This parameter reflects the frequency
and/or amplitude of [Ca2+]i oscillations and
is largely independent of the actual [Ca2+]i
value (18, 19).
Statistics
A two-way ANOVA was employed to analyze the data. Differences in
mean levels of PRL gene expression, mean
[Ca2+]i values, and oscillation indexes for
different groups of cells were compared by use of Bonferronis
multiple comparisons test. Differences were considered significant at
P < 0.05.
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to E. E. Glavé for expert technical
assistance, and to Dr. L. Núñez for help in analysis of
calcium oscillations. We thank J. Nicholson (Pathology Department,
Medical University of South Carolina) for access to the Attofluor Ratio
Vision System. We also thank Drs. E. J. Abraham, K. D.
Nusser, and S. T. Willard as well as Ms. A. Gore for critique of
the manuscript. We are grateful to Dr. R Maurer for the gift of the
rPRL-LUC plasmid.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. L. Stephen Frawley, Laboratory of Molecular Dynamics. Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, South Carolina 29425.
This work was supported by NIH Grant DK-38215 (to L.S.F.) and a
postdoctoral fellowship from the Ministerio de Educación y
Cultura of Spain (to C.V.).
Received for publication September 8, 1997.
Revision received October 17, 1997.
Accepted for publication October 27, 1997.
 |
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