0013-7227/90/1272-0613$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society
Vol. 127, No. 2 Printed in U.S.A.
Calcitonin Inhibits Thyrotropin-Releasing Hormone Induced Increases in Cytosolic Ca2+ in Isolated Rat Anterior Pituitary Cells* G. V. SHAH, D. KENNEDY, M. E. DOCKTER, AND W. R. CROWLEY Departments of Pharmacology (G. V.S., W.R.C.), Microbiology and Immunology (D.K., M.E.D.), and Medicine (M.E.D.), University of Tennessee-Memphis College of Medicine, Memphis, Tennessee 38163 during the scans, and 100% of the cells responded to the Ca2+ ionophore ionomycin with increases in the Indo-1 ratio. Approximately 25-30% of the AP cells responded to a 1 juM pulse of TRH with marked increases in the Indo-1 ratio, indicative of increases in [Ca2+]i, with the response consisting of two phases, an initial rapid rise that was unaffected by the presence of EGTA in the extracellular environment, followed by a decrease to a sustained secondary phase that was completely eliminated by EGTA. In a normal extracellular Ca2+ environment, pretreatment with 100 nM sCT almost totally inhibited the response to 1 fiU TRH. In EGTA-pretreated AP cells, the initial EGTAinsensitive phase of the TRH-induced [Ca2+]i increase was also abolished by prior exposure to sCT. These results suggest that sCT inhibits TRH-stimulated PRL release in AP cells by attenuating the TRH-induced increase in [Ca2+]i, an effect that probably occurs as a consequence of inhibition of the stimulatory effect of TRH on the Ca2+/phospholipid messenger system. (Endocrinology 127: 613-620, 1990)
ABSTRACT. Calcitonin (CT) and related peptides, such as CT gene-related peptide and salmon CT (sCT)-like peptide, are present in the rat nervous system and the pituitary gland, and sCT markedly inhibits basal and TRH-stimulated PRL release from anterior pituitary (AP) cells. Because TRH-induced PRL release is known to involve increases in cytosolic free Ca2+ derived from both extracellular and intracellular sources, the objective of the present study was to test whether sCT interferes with this effect. Secretogogue-induced elevations of cytosolic free Ca2+ ([Ca2+]i) in acutely dispersed AP cells were monitored using the fluorescent Ca2+ indicator Indo-1 AM and flow cytometry. AP cells were enzymatically dispersed to single cell suspensions and loaded with 20 pM Indo-1 AM for 30 min. Indo-1loaded AP cells were scanned at a rate of approximately 500 cells/sec for 200-300 sec in a flow cytometer, and the ratio of fluorescence due to Ca2+ bound to Indo-1 to free Indo-1 (Indo-1 ratio), which is an index of [Ca2+]i, was determined for each cell. Under basal conditions, AP cells showed stable Indo-1 ratios
T
detected in rat brain and the pituitary gland (9-10), specifically and competitively inhibits TRH-stimulated PRL release from cultured AP cells. Further studies with perifused AP cells (8) suggest that sCT markedly inhibits both phases of TRH-stimulated PRL release, with a particularly marked effect on the early [Ca2+]i/phospholipid-dependent "spike" phase. Moreover, because sCT antagonizes PRL release induced by TRH, but not by high concentrations of the Ca2+ ionophore A23187, sCT appears to affect events leading to, rather than subsequent to, the increase in [ Ca2+]i (8). Effects of sCT, and its possible interaction with TRH, on the levels of [Ca2+]i in AP cells have not been reported. Methods to measure the changes in [Ca2+]i levels include monitoring the fluorescence of intracellularly trapped Ca2+-chelators such as Quin-2 or Fura-2 (11). These methods can be used at the single cell level, but when used to monitor secretogogue-induced changes in cytoplasmic Ca2+ levels in a large population of cells, measure an overall level of fluorescence of all the cells in a sample. In a sample of heterogeneous cells, such as primary AP cells, this may underestimate the changes in
HE BINDING of factors such as angiotensin II and TRH to their plasma membrane receptors on anterior pituitary (AP) lactotrophe cells causes a rapid increase in cytoplasmic free calcium ion concentrations ([Ca2+]i), which subsequently stimulates a variety of cellular processes, ultimately leading to PRL release (1, 2). These events are associated with the degradation of phosphoinositol 4,5 bisphosphate (PIP2) in the cell membrane to diacyglycerol and inositol 1,4,5-trisphosphate (IP3). Recent evidence indicates that IP 3 increases cytoplasmic [Ca2+]i, predominantly by redistribution of Ca2+ from intracellular stores, while diacylglycerol activates protein kinase C, and leads to the influx of Ca2+ through the voltage-gated Ca2+ channels (1-4). Our previous studies (5-7), as well as the findings presented in a companion study (8), demonstrate that salmon calcitonin (sCT)-like peptide, which has been Received March 2, 1990. Address all correspondence and requests for reprints to: Dr. William R. Crowley, Department of Pharmacology, University of Tennessee— Memphis, Memphis, Tennessee 38163. * This work was supported by NIH Grant HD-13703 (to W.R.C.).
613
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 00:30 For personal use only. No other uses without permission. . All rights reserved.
CT INHIBITS PITUITARY Ca2+
614
[Ca2+]i of the responding cell subpopulations. The technique of ratio flow cytometry can examine the changes in the cytosolic [Ca2+]i at the level of an individual cell while simultaneously scanning the overall population, and any changes can be quantitated in terms of the overall changes in the population mean [Ca2+]i levels, as well as in any responding subpopulation. The recently developed fluorescent Ca2+-chelator, Indo-1 AM, has the properties required for flow cytometric measurements of [Ca2+]i at the single cell level (12). When internalized Indo-1 binds free Ca2+, and is excited near the optimal wavelength of 356 nm, its fluorescence emission peak shifts from 486 nm, for the free dye, to 404 nm, for the Ca2+/Indo-1 complex, with minimal change in the total fluorescence intensity (12). The ratio of fluorescence emission at these two wavelengths (404/486, the Indo-1 ratio) thus reflects the fraction of Ca2+-bound dye molecules and provides a fully objective and precise measurement of cytoplasmic [Ca2+]i that is independent of the individual cell's size or its dye content (13, 14). The objectives of the present study were to examine 1) whether Indo-1 ratio flow cytometry can detect TRHinduced changes in the cytosolic [Ca2+]i in dispersed AP cells, and 2) to test the hypothesis that sCT inhibits TRH-stimulated PRL release by attenuating the TRHinduced increase in cytoplasmic [Ca2+]i in AP cells.
Materials and Methods Animals Sixty-day-old Holtzman female rats (Sasco Laboratories, Omaha, NE) were ovariectomized and, after 1-2 weeks of recovery, were implanted sc with 20 mm long Silastic capsules (i.d. 1.5 mm, Dow Corning, Midland, MI) filled with 250 ng estradiol in sesame oil for 3 days. Such capsules result in serum estradiol levels similar to that seen on proestrus (60-100 pg/ ml) (15). The rats were sacrificed by decapitation and the AP glands were collected. Preparation and labeling of AP cells with Indo-1 AM AP cells were prepared by enzymatic dispersion of the pituitary glands as previously described (5). The cells were suspended in Dulbecco's modified Eagle's medium supplemented with BSA (3 g/1), HEPES (10 rail), bacitracin (20 /ZM), and gentamicin (10 mg/liter), and were incubated with 20 /*M Indo1 AM at 37 C for 30 min. The Indo-1-labeled cells were then diluted 1:10 with Dulbecco's modified Eagle's medium, which provides an extracellular Ca2+ concentration of 1.6 mM, and subjected to flow cytometry. Cells were maintained at 37 C until loading into the sample chamber of the flow cytometer. Flow cytometric analysis The analysis was performed on a Coulter Corporation EPICS 753 flow cytometer. The front 5 Watt argon ion laser of this instrument was used in the ultraviolet mode (351-364 nm) at
Endo • 1990 Vol 127-No 2
a constant power of 100 milliWatts. The Indo-1 labeled AP cells were scanned at an approximate rate of 500 cells/sec for a total of 200, or in some experiments, 300 sec, at room temperature. Three signals were measured as each cell passed through the instrument. Forward angle light scatter was measured using a linear signal without the use of a neutral density filter in front of the detector. The fluorescence signals produced by the internalized Indo-1 were measured on two separate detectors after splitting the signal with a 453-nm dichroic mirror (MicroCoatings, Inc., Westford, MA). The shorter wavelengths were measured as linear integrated signals after passing through a 400-nm bandpass filter (MicroCoatings, Inc., Westford, MA). This signal is predominantly the fluorescence of the Ca2+-Indo-1 complex. The longer wavelengths were measured after passing through a 486-nm bandpass filter (MicroCoatings, Inc., Westford, MA). This fluorescence is that produced predominantly from the Ca2+-free form of internalized Indo-1. Two additional parameters, the time and the ratio of fluorescence signal at 404 nm and at 486 nm were generated for each cell during each set of measurements. This ratio was generated from the instrument's analog ratio circuitry, which was gated on the 486-nm signal to exclude instrument noise and debris seen in lower fluorescence channels and clumps of cells at the higher fluorescence channels. A standard of Hoechst 33342 stained alignment micro beads (Flow Cytometry Standards Corporation, Research Triangle Park, NC) was run before each set of experiments to calibrate fluorescence intensity and to obtain a coefficient of variation on the fluorescence measurement. For each individual experiment, baseline data from unstimulated cells were collected for 35 sec before addition of the test hormone or agent. In order to continuously monitor hormone-induced changes in [Ca2+]i levels, the test hormone or agent was injected directly into the pressurized sample chamber with a Hamilton microsyringe while scanning was ongoing. Transit time from the sample chamber through the tubing to the interrogation point of the instrument was 15 sec. The data from stimulated cells were then collected for the remainder of the 200-sec period scan. Data analysis Data were analyzed on a Coulter Corporation EASY 2 remote data analysis station running EASY 2 flow cytometric analysis software (Coulter Corporation, Hialeah, FL). Representative individual experiments are depicted in three parameter scattergrams generated using the CUBE program and plotted on a Hewlett Packard 7550A plotter. These plots show both the ratio of Indo-1 fluorescence (404/486) per cell on the y-axis, and the number of cells (frequency, depicted in different shades) per time channel plotted against scanning time on the x-axis. The frequency of AP cells per time channel is presented in the scattergrams in three regions (HE), (•) and (H), which represent 70%, 25%, and 5%, respectively, of total AP cells/ time channel. To evaluate the effect of TRH and sCT on Ca2+, the data are analyzed and presented as: 1) mean Indo-1 fluorescence ratio (404/486) over time of all the pituitary cells (including non-responding cells), which corresponds to the overall population mean [Ca2+]i, and 2) as the net proportion or number of responding cells (i.e., those cells having a fluores-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 00:30 For personal use only. No other uses without permission. . All rights reserved.
CT INHIBITS PITUITARY Ca2+ cence ratio above the defined threshold levels) over time. This profile is essentially equivalent to the changes in Indo-1 ratio in the responding subpopulation. The threshold level was defined as that range shown by the highest 5% of cells during the baseline period. Thus, during a control (unstimulated) scan, 95% of AP cells will show Indo-1 fluorescence ratios below this level, and a cell responding to a secretagogue, therefore, is defined as one in which the fluorescence ratio during the stimulation period was at or above the threshold level. The net response was corrected for the 5% baseline. The data from the replicate experiments were pooled and are presented as mean ± SEM. The areas of fluorescence ratio under the TRH and (sCT + TRH) response curves were digitized using a Summagraphics digitizer interfaced with an IBM PC XT computer and the SigmaScan software (Jandel Scientific Co., Corte Madera, CA). Differences in these values between control (TRH alone) and experimental (sCT + TRH) groups were tested with unpaired Student's t-tests.
Results 2+
Measurement of [Ca ]i of AP cells by flow effects of EGTA and ionomycin
cytometry:
Figure 1 depicts resting Indo-1 ratios during a typical control scan in the three parameter scattergram format. Acutely dispersed AP cells had widely distributed values of [Ca2+]i, expressed as the Indo-1 fluorescence ratio (mean = 0.281; half height coefficient of variation or HCV, which indicates the spread of resting [Ca2+]i levels, = 0.313; Fig 1, left panel). When suspended in a medium with low [Ca2+] and 2 mM EGTA, AP cells showed a decline in the Indo-1 ratio, as well as in the width of
615
their distribution (mean = 0.234; HCV = 0.270) (Fig 1, right panel). As shown in the representative scans in Fig 1, in the absence of experimental manipulations, the cells exhibited relatively stable baseline fluorescence during a scan of 200 sec under either of these conditions. Figure 2 shows that after treatment with the Ca2+ ionophore ionomycin (20 ixM), in order to cause the Ca2+ in the media to flood into the cells, there was rapid increase in the Indo-1 ratio, indicative of increased [Ca2+]i in all of the AP cells. Indo-1 ratio values reached a maximum within a few seconds, and remained at this level until the end of the scan. Effects of TRH in the absence and presence of EGTA The left panel of Fig 3 presents a typical TRH-induced change in Indo-1 ratios of AP cells during a scan of 300 sec in the three-parameter scattergram format. TRH (1 ;UM) caused a transient increase in the Indo-1 ratios in approximately 25-30% of the AP cells. The responding population showed a rapid, sharp, and brief rise in the fluorescence ratio that peaked within approximately 9 sec, and then declined to its lowest level within 80 sec after reaching the maximum. A subsequent secondary phase in which the mean fluorescence ratios were maintained at a lower level, but still higher than prestimulation baseline, was observed from 127 sec onward (left 60
00 ^
50-
6B
40
1 CO
DC G)
u
3
c a> o
IB
W 10
20
30
40
50
Time (seconds x 3)
60
10
20
30
40
50
60
10-
Time (seconds x 3)
FIG. 1. Resting Indo-1 ratios in AP cells. The left panel is a three parameter scattergram where both the Indo-1 ratio per cell and the number of cells per channel (in three shades) are shown over a 200-sec scan, and is indicative of the distribution of resting [Ca2+]i levels in a mixed population of AP cells suspended in a DMEM containing 1.6 mM Ca2+. The percentage of AP cells per time channel in this and in the subsequent three parameter scattergram figures is presented in three regions encoded as (Id), (•) and (•), which represent 70%, 25%, and 5%, respectively, of total AP cells. Also, in this and subsequent figures, the Indo-1 ratio values on the y-axis have been multiplied by 64 by the analytical software. The right panel shows the typical Indo1 ratio distribution in AP cells suspended in a medium containing low Ca2+ (0.16 mM) + 2 mM EGTA, over a 200-sec scan.
U.
10
50
60
Time (seconds x 3) FIG. 2. Effect of ionomycin on Indo-1 ratios in AP cells. The three parameter scattergram shows an example of the effect of ionomycin (20 HM), which was added at the time channel 11. This causes the Ca2+ (1.6 mM) in the media to flood into the cells, resulting in a rapid increase in [Ca2+]i levels as indicated by the increase in the Indo-1 ratio for time periods between channels 12 and 63 (time period 57 sec to 200 sec).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 00:30 For personal use only. No other uses without permission. . All rights reserved.
616
CT INHIBITS PITUITARY Ca2+ FIG. 3. Effect of TRH on [Ca2+]i levels in AP cells. The left panel is a three parameter scattergram showing a representative change in Indo-1 ratios of AP cells in response to TRH. Addition of 1 nM TRH at time channel 8 causes a transient and biphasic increase in a subpopulation of AP cells. The right panel presents the typical TRH response in AP cells that were pretreated with 100 nM sCT for 5 min prior to the addition of TRH. Only a small number of sCT pretreated AP cells show an increase in Indo-1 ratio in response to 1 /uM TRH.
2.
Endo-1990 Voll27-No2
SB
10
20
38
40
50
10,.
60
panel of Fig. 3). When TRH was applied to AP cells in the presence of 2 mM EGTA, the initial Indo-1 fluorescence peak was clearly observed, but the subsequent secondary response was absent (left panel of Fig 4). Data from several replicate experiments showing the mean ± SEM responses to TRH in the presence and absence of EGTA are presented in Fig 5. These data are presented in terms of the number of AP cells showing Indo-1 fluorescence ratios above threshold levels during the 300 sec scan, and thus show the pattern of change in the responding subpopulation (25-30% of the total). Figure 5 shows clearly in the control cells the presence of a marked initial rise, to a level approximately 3-fold higher than baseline, followed by a prolonged period in which the Indo-1 ratios were maintained approximately 30% above the prestimulation baseline {P < 0.01 vs. prestimulation baseline, based on repeated measures analysis of variance). The earlier phase of the response was not affected by the presence of EGTA in the extracellular environment, but the delayed phase of the TRH response was eliminated (Fig. 5, inset), suggesting this component of the response may be dependent upon extracellular Ca2+. Figure 6 presents grouped data of mean Indo-1 fluorescence ratios from replicate experiments with control
30 .40
50
(n= 1 replicate, 200-sec scans) and sCT-treated cells (n = 7 scans). The results indicate that the overall population mean Indo-1 fluorescence ratio of the control cells increased from 0.313 to 0.422 within the first 9 sec of TRH stimulation. After reaching this peak, the mean ratio rapidly declined to 0.358 in next 30 sec, and thereafter declined at a much slower rate to the prestimulation resting levels by approximately 111 sec after TRH stimulation. This may be contrasted with the response of control cells to TRH depicted in Fig 7, in which all cells were pretreated for 60 sec with 2 mM EGTA. Such treatment decreased resting Indo-1 ratio values. The rapid increase in fluorescence ratios after TRH is still apparent, but the population mean Indo-1 ratios declined much more rapidly and reached prestimulation baseline within 65 sec. Effect of sCT on the response to TRH The right panel of Fig 3 shows an individual example of the effect of sCT (100 nM) on the Indo-1 ratio response to TRH in normal cells. When added to AP cells 5 min prior to TRH (1 /xM), SCT caused a substantial inhibition in the TRH-induced increase in the Indo-1 fluorescence ratio. Figure 6 presents grouped data from 7 replicate
60i
S
50-
50-
40-
30,,11111
FlG. 4. Effect of TRH on Indo-1 ratios in AP cells in the presence of 2 mM EGTA. The left panel is a three parameter scattergram showing a representative TRH-induced change in Indo-1 ratio of AP cells in medium with normal Ca2+. Addition of 2 mM EGTA at time channel 2 causes a gradual decrease in the ratios, which stabilize by time channel 16. Addition of 1 nM TRH at time channel 23 causes a transient increase in a subpopulation of AP cells. The right panel shows a similar experiment in AP cells that were pretreated with 100 nM sCT for 5 min prior to addition of TRH. sCT almost totally blocks the effect of TRH.
20
Time (seconds x 4.7)
Time (seconds x 4.7)
2
20- I
20•
10-
10-
10
20
30
40
50
Time (seconds x 4.7)
§i™l I l l l IIIHIIfJlfllMIII III Illllllllllh 10
20
30
40
50
Time (seconds x 4.7)
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 00:30 For personal use only. No other uses without permission. . All rights reserved.
60
CT INHIBITS PITUITARY Ca2+
617
1000 200 •
X•2
^V
g 600 n
>
^
20
30
40
S S
15--
200 >•• • i
0
10
20
20
30
Time Channel (seconds x 4.7) FIG. 5. Effects of EGTA on the number of cells responding to TRH. The plot shows responses to 1 nM TRH in the presence and absence of 2 mM EGTA from three replicate experiments in each group. The mean ± SEM number of cells exhibiting fluorescence above threshold in the absence of EGTA ( • D) and in presence of EGTA ( • • ) , which was added at the start of the scan, are depicted. The inset highlights the difference in the groups during latter part of the responses (secondary phase). Note that the standard error bars are frequently smaller than the mean symbols. 28 A
«
26--
CO
5 o S
TRH
24-
ij
22
j
40
50
60
FlG. 7. Effect of sCT on TRH-induced increases in Indo-1 ratios in EGTA-treated AP cells. The plot presents the overall population mean ± SEM Indo-1 ratio data from 7 replicate 300-sec scans in each condition. Both groups of cells were exposed to 2 mM EGTA at time channel 9 and then to 1 nM TRH (=) at time channel 21. Control cells (A A) show a transient increase in Indo-1 ratio in response to TRH, while a 5-min pretreatment with 100 nM sCT ( ,• • ) significantly inhibited the overall Indo-1 ratio response (P < 0.001, unpaired t-tests on digitized areas under the curves).
to 3-5% (P < 0.001, t-test). sCT also significantly curtailed the brief initial Indo1 ratio response to TRH in cells treated with 2 mM EGTA, as shown in the right panel of Fig 4 as a representative individual example, and in Fig 7, depicting group means ± SEM from 7 scans per treatment condition. In these studies, the mean Indo-1 ratios were reduced by the sCT treatment, and then further reduced by EGTA as expected, prior to the addition of TRH.
TJTTTT
Discussion
20
Vol. 127, No. 2 Printed in U.S.A.
Calcitonin Inhibits Thyrotropin-Releasing Hormone Induced Increases in Cytosolic Ca2+ in Isolated Rat Anterior Pituitary Cells* G. V. SHAH, D. KENNEDY, M. E. DOCKTER, AND W. R. CROWLEY Departments of Pharmacology (G. V.S., W.R.C.), Microbiology and Immunology (D.K., M.E.D.), and Medicine (M.E.D.), University of Tennessee-Memphis College of Medicine, Memphis, Tennessee 38163 during the scans, and 100% of the cells responded to the Ca2+ ionophore ionomycin with increases in the Indo-1 ratio. Approximately 25-30% of the AP cells responded to a 1 juM pulse of TRH with marked increases in the Indo-1 ratio, indicative of increases in [Ca2+]i, with the response consisting of two phases, an initial rapid rise that was unaffected by the presence of EGTA in the extracellular environment, followed by a decrease to a sustained secondary phase that was completely eliminated by EGTA. In a normal extracellular Ca2+ environment, pretreatment with 100 nM sCT almost totally inhibited the response to 1 fiU TRH. In EGTA-pretreated AP cells, the initial EGTAinsensitive phase of the TRH-induced [Ca2+]i increase was also abolished by prior exposure to sCT. These results suggest that sCT inhibits TRH-stimulated PRL release in AP cells by attenuating the TRH-induced increase in [Ca2+]i, an effect that probably occurs as a consequence of inhibition of the stimulatory effect of TRH on the Ca2+/phospholipid messenger system. (Endocrinology 127: 613-620, 1990)
ABSTRACT. Calcitonin (CT) and related peptides, such as CT gene-related peptide and salmon CT (sCT)-like peptide, are present in the rat nervous system and the pituitary gland, and sCT markedly inhibits basal and TRH-stimulated PRL release from anterior pituitary (AP) cells. Because TRH-induced PRL release is known to involve increases in cytosolic free Ca2+ derived from both extracellular and intracellular sources, the objective of the present study was to test whether sCT interferes with this effect. Secretogogue-induced elevations of cytosolic free Ca2+ ([Ca2+]i) in acutely dispersed AP cells were monitored using the fluorescent Ca2+ indicator Indo-1 AM and flow cytometry. AP cells were enzymatically dispersed to single cell suspensions and loaded with 20 pM Indo-1 AM for 30 min. Indo-1loaded AP cells were scanned at a rate of approximately 500 cells/sec for 200-300 sec in a flow cytometer, and the ratio of fluorescence due to Ca2+ bound to Indo-1 to free Indo-1 (Indo-1 ratio), which is an index of [Ca2+]i, was determined for each cell. Under basal conditions, AP cells showed stable Indo-1 ratios
T
detected in rat brain and the pituitary gland (9-10), specifically and competitively inhibits TRH-stimulated PRL release from cultured AP cells. Further studies with perifused AP cells (8) suggest that sCT markedly inhibits both phases of TRH-stimulated PRL release, with a particularly marked effect on the early [Ca2+]i/phospholipid-dependent "spike" phase. Moreover, because sCT antagonizes PRL release induced by TRH, but not by high concentrations of the Ca2+ ionophore A23187, sCT appears to affect events leading to, rather than subsequent to, the increase in [ Ca2+]i (8). Effects of sCT, and its possible interaction with TRH, on the levels of [Ca2+]i in AP cells have not been reported. Methods to measure the changes in [Ca2+]i levels include monitoring the fluorescence of intracellularly trapped Ca2+-chelators such as Quin-2 or Fura-2 (11). These methods can be used at the single cell level, but when used to monitor secretogogue-induced changes in cytoplasmic Ca2+ levels in a large population of cells, measure an overall level of fluorescence of all the cells in a sample. In a sample of heterogeneous cells, such as primary AP cells, this may underestimate the changes in
HE BINDING of factors such as angiotensin II and TRH to their plasma membrane receptors on anterior pituitary (AP) lactotrophe cells causes a rapid increase in cytoplasmic free calcium ion concentrations ([Ca2+]i), which subsequently stimulates a variety of cellular processes, ultimately leading to PRL release (1, 2). These events are associated with the degradation of phosphoinositol 4,5 bisphosphate (PIP2) in the cell membrane to diacyglycerol and inositol 1,4,5-trisphosphate (IP3). Recent evidence indicates that IP 3 increases cytoplasmic [Ca2+]i, predominantly by redistribution of Ca2+ from intracellular stores, while diacylglycerol activates protein kinase C, and leads to the influx of Ca2+ through the voltage-gated Ca2+ channels (1-4). Our previous studies (5-7), as well as the findings presented in a companion study (8), demonstrate that salmon calcitonin (sCT)-like peptide, which has been Received March 2, 1990. Address all correspondence and requests for reprints to: Dr. William R. Crowley, Department of Pharmacology, University of Tennessee— Memphis, Memphis, Tennessee 38163. * This work was supported by NIH Grant HD-13703 (to W.R.C.).
613
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 00:30 For personal use only. No other uses without permission. . All rights reserved.
CT INHIBITS PITUITARY Ca2+
614
[Ca2+]i of the responding cell subpopulations. The technique of ratio flow cytometry can examine the changes in the cytosolic [Ca2+]i at the level of an individual cell while simultaneously scanning the overall population, and any changes can be quantitated in terms of the overall changes in the population mean [Ca2+]i levels, as well as in any responding subpopulation. The recently developed fluorescent Ca2+-chelator, Indo-1 AM, has the properties required for flow cytometric measurements of [Ca2+]i at the single cell level (12). When internalized Indo-1 binds free Ca2+, and is excited near the optimal wavelength of 356 nm, its fluorescence emission peak shifts from 486 nm, for the free dye, to 404 nm, for the Ca2+/Indo-1 complex, with minimal change in the total fluorescence intensity (12). The ratio of fluorescence emission at these two wavelengths (404/486, the Indo-1 ratio) thus reflects the fraction of Ca2+-bound dye molecules and provides a fully objective and precise measurement of cytoplasmic [Ca2+]i that is independent of the individual cell's size or its dye content (13, 14). The objectives of the present study were to examine 1) whether Indo-1 ratio flow cytometry can detect TRHinduced changes in the cytosolic [Ca2+]i in dispersed AP cells, and 2) to test the hypothesis that sCT inhibits TRH-stimulated PRL release by attenuating the TRHinduced increase in cytoplasmic [Ca2+]i in AP cells.
Materials and Methods Animals Sixty-day-old Holtzman female rats (Sasco Laboratories, Omaha, NE) were ovariectomized and, after 1-2 weeks of recovery, were implanted sc with 20 mm long Silastic capsules (i.d. 1.5 mm, Dow Corning, Midland, MI) filled with 250 ng estradiol in sesame oil for 3 days. Such capsules result in serum estradiol levels similar to that seen on proestrus (60-100 pg/ ml) (15). The rats were sacrificed by decapitation and the AP glands were collected. Preparation and labeling of AP cells with Indo-1 AM AP cells were prepared by enzymatic dispersion of the pituitary glands as previously described (5). The cells were suspended in Dulbecco's modified Eagle's medium supplemented with BSA (3 g/1), HEPES (10 rail), bacitracin (20 /ZM), and gentamicin (10 mg/liter), and were incubated with 20 /*M Indo1 AM at 37 C for 30 min. The Indo-1-labeled cells were then diluted 1:10 with Dulbecco's modified Eagle's medium, which provides an extracellular Ca2+ concentration of 1.6 mM, and subjected to flow cytometry. Cells were maintained at 37 C until loading into the sample chamber of the flow cytometer. Flow cytometric analysis The analysis was performed on a Coulter Corporation EPICS 753 flow cytometer. The front 5 Watt argon ion laser of this instrument was used in the ultraviolet mode (351-364 nm) at
Endo • 1990 Vol 127-No 2
a constant power of 100 milliWatts. The Indo-1 labeled AP cells were scanned at an approximate rate of 500 cells/sec for a total of 200, or in some experiments, 300 sec, at room temperature. Three signals were measured as each cell passed through the instrument. Forward angle light scatter was measured using a linear signal without the use of a neutral density filter in front of the detector. The fluorescence signals produced by the internalized Indo-1 were measured on two separate detectors after splitting the signal with a 453-nm dichroic mirror (MicroCoatings, Inc., Westford, MA). The shorter wavelengths were measured as linear integrated signals after passing through a 400-nm bandpass filter (MicroCoatings, Inc., Westford, MA). This signal is predominantly the fluorescence of the Ca2+-Indo-1 complex. The longer wavelengths were measured after passing through a 486-nm bandpass filter (MicroCoatings, Inc., Westford, MA). This fluorescence is that produced predominantly from the Ca2+-free form of internalized Indo-1. Two additional parameters, the time and the ratio of fluorescence signal at 404 nm and at 486 nm were generated for each cell during each set of measurements. This ratio was generated from the instrument's analog ratio circuitry, which was gated on the 486-nm signal to exclude instrument noise and debris seen in lower fluorescence channels and clumps of cells at the higher fluorescence channels. A standard of Hoechst 33342 stained alignment micro beads (Flow Cytometry Standards Corporation, Research Triangle Park, NC) was run before each set of experiments to calibrate fluorescence intensity and to obtain a coefficient of variation on the fluorescence measurement. For each individual experiment, baseline data from unstimulated cells were collected for 35 sec before addition of the test hormone or agent. In order to continuously monitor hormone-induced changes in [Ca2+]i levels, the test hormone or agent was injected directly into the pressurized sample chamber with a Hamilton microsyringe while scanning was ongoing. Transit time from the sample chamber through the tubing to the interrogation point of the instrument was 15 sec. The data from stimulated cells were then collected for the remainder of the 200-sec period scan. Data analysis Data were analyzed on a Coulter Corporation EASY 2 remote data analysis station running EASY 2 flow cytometric analysis software (Coulter Corporation, Hialeah, FL). Representative individual experiments are depicted in three parameter scattergrams generated using the CUBE program and plotted on a Hewlett Packard 7550A plotter. These plots show both the ratio of Indo-1 fluorescence (404/486) per cell on the y-axis, and the number of cells (frequency, depicted in different shades) per time channel plotted against scanning time on the x-axis. The frequency of AP cells per time channel is presented in the scattergrams in three regions (HE), (•) and (H), which represent 70%, 25%, and 5%, respectively, of total AP cells/ time channel. To evaluate the effect of TRH and sCT on Ca2+, the data are analyzed and presented as: 1) mean Indo-1 fluorescence ratio (404/486) over time of all the pituitary cells (including non-responding cells), which corresponds to the overall population mean [Ca2+]i, and 2) as the net proportion or number of responding cells (i.e., those cells having a fluores-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 00:30 For personal use only. No other uses without permission. . All rights reserved.
CT INHIBITS PITUITARY Ca2+ cence ratio above the defined threshold levels) over time. This profile is essentially equivalent to the changes in Indo-1 ratio in the responding subpopulation. The threshold level was defined as that range shown by the highest 5% of cells during the baseline period. Thus, during a control (unstimulated) scan, 95% of AP cells will show Indo-1 fluorescence ratios below this level, and a cell responding to a secretagogue, therefore, is defined as one in which the fluorescence ratio during the stimulation period was at or above the threshold level. The net response was corrected for the 5% baseline. The data from the replicate experiments were pooled and are presented as mean ± SEM. The areas of fluorescence ratio under the TRH and (sCT + TRH) response curves were digitized using a Summagraphics digitizer interfaced with an IBM PC XT computer and the SigmaScan software (Jandel Scientific Co., Corte Madera, CA). Differences in these values between control (TRH alone) and experimental (sCT + TRH) groups were tested with unpaired Student's t-tests.
Results 2+
Measurement of [Ca ]i of AP cells by flow effects of EGTA and ionomycin
cytometry:
Figure 1 depicts resting Indo-1 ratios during a typical control scan in the three parameter scattergram format. Acutely dispersed AP cells had widely distributed values of [Ca2+]i, expressed as the Indo-1 fluorescence ratio (mean = 0.281; half height coefficient of variation or HCV, which indicates the spread of resting [Ca2+]i levels, = 0.313; Fig 1, left panel). When suspended in a medium with low [Ca2+] and 2 mM EGTA, AP cells showed a decline in the Indo-1 ratio, as well as in the width of
615
their distribution (mean = 0.234; HCV = 0.270) (Fig 1, right panel). As shown in the representative scans in Fig 1, in the absence of experimental manipulations, the cells exhibited relatively stable baseline fluorescence during a scan of 200 sec under either of these conditions. Figure 2 shows that after treatment with the Ca2+ ionophore ionomycin (20 ixM), in order to cause the Ca2+ in the media to flood into the cells, there was rapid increase in the Indo-1 ratio, indicative of increased [Ca2+]i in all of the AP cells. Indo-1 ratio values reached a maximum within a few seconds, and remained at this level until the end of the scan. Effects of TRH in the absence and presence of EGTA The left panel of Fig 3 presents a typical TRH-induced change in Indo-1 ratios of AP cells during a scan of 300 sec in the three-parameter scattergram format. TRH (1 ;UM) caused a transient increase in the Indo-1 ratios in approximately 25-30% of the AP cells. The responding population showed a rapid, sharp, and brief rise in the fluorescence ratio that peaked within approximately 9 sec, and then declined to its lowest level within 80 sec after reaching the maximum. A subsequent secondary phase in which the mean fluorescence ratios were maintained at a lower level, but still higher than prestimulation baseline, was observed from 127 sec onward (left 60
00 ^
50-
6B
40
1 CO
DC G)
u
3
c a> o
IB
W 10
20
30
40
50
Time (seconds x 3)
60
10
20
30
40
50
60
10-
Time (seconds x 3)
FIG. 1. Resting Indo-1 ratios in AP cells. The left panel is a three parameter scattergram where both the Indo-1 ratio per cell and the number of cells per channel (in three shades) are shown over a 200-sec scan, and is indicative of the distribution of resting [Ca2+]i levels in a mixed population of AP cells suspended in a DMEM containing 1.6 mM Ca2+. The percentage of AP cells per time channel in this and in the subsequent three parameter scattergram figures is presented in three regions encoded as (Id), (•) and (•), which represent 70%, 25%, and 5%, respectively, of total AP cells. Also, in this and subsequent figures, the Indo-1 ratio values on the y-axis have been multiplied by 64 by the analytical software. The right panel shows the typical Indo1 ratio distribution in AP cells suspended in a medium containing low Ca2+ (0.16 mM) + 2 mM EGTA, over a 200-sec scan.
U.
10
50
60
Time (seconds x 3) FIG. 2. Effect of ionomycin on Indo-1 ratios in AP cells. The three parameter scattergram shows an example of the effect of ionomycin (20 HM), which was added at the time channel 11. This causes the Ca2+ (1.6 mM) in the media to flood into the cells, resulting in a rapid increase in [Ca2+]i levels as indicated by the increase in the Indo-1 ratio for time periods between channels 12 and 63 (time period 57 sec to 200 sec).
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 00:30 For personal use only. No other uses without permission. . All rights reserved.
616
CT INHIBITS PITUITARY Ca2+ FIG. 3. Effect of TRH on [Ca2+]i levels in AP cells. The left panel is a three parameter scattergram showing a representative change in Indo-1 ratios of AP cells in response to TRH. Addition of 1 nM TRH at time channel 8 causes a transient and biphasic increase in a subpopulation of AP cells. The right panel presents the typical TRH response in AP cells that were pretreated with 100 nM sCT for 5 min prior to the addition of TRH. Only a small number of sCT pretreated AP cells show an increase in Indo-1 ratio in response to 1 /uM TRH.
2.
Endo-1990 Voll27-No2
SB
10
20
38
40
50
10,.
60
panel of Fig. 3). When TRH was applied to AP cells in the presence of 2 mM EGTA, the initial Indo-1 fluorescence peak was clearly observed, but the subsequent secondary response was absent (left panel of Fig 4). Data from several replicate experiments showing the mean ± SEM responses to TRH in the presence and absence of EGTA are presented in Fig 5. These data are presented in terms of the number of AP cells showing Indo-1 fluorescence ratios above threshold levels during the 300 sec scan, and thus show the pattern of change in the responding subpopulation (25-30% of the total). Figure 5 shows clearly in the control cells the presence of a marked initial rise, to a level approximately 3-fold higher than baseline, followed by a prolonged period in which the Indo-1 ratios were maintained approximately 30% above the prestimulation baseline {P < 0.01 vs. prestimulation baseline, based on repeated measures analysis of variance). The earlier phase of the response was not affected by the presence of EGTA in the extracellular environment, but the delayed phase of the TRH response was eliminated (Fig. 5, inset), suggesting this component of the response may be dependent upon extracellular Ca2+. Figure 6 presents grouped data of mean Indo-1 fluorescence ratios from replicate experiments with control
30 .40
50
(n= 1 replicate, 200-sec scans) and sCT-treated cells (n = 7 scans). The results indicate that the overall population mean Indo-1 fluorescence ratio of the control cells increased from 0.313 to 0.422 within the first 9 sec of TRH stimulation. After reaching this peak, the mean ratio rapidly declined to 0.358 in next 30 sec, and thereafter declined at a much slower rate to the prestimulation resting levels by approximately 111 sec after TRH stimulation. This may be contrasted with the response of control cells to TRH depicted in Fig 7, in which all cells were pretreated for 60 sec with 2 mM EGTA. Such treatment decreased resting Indo-1 ratio values. The rapid increase in fluorescence ratios after TRH is still apparent, but the population mean Indo-1 ratios declined much more rapidly and reached prestimulation baseline within 65 sec. Effect of sCT on the response to TRH The right panel of Fig 3 shows an individual example of the effect of sCT (100 nM) on the Indo-1 ratio response to TRH in normal cells. When added to AP cells 5 min prior to TRH (1 /xM), SCT caused a substantial inhibition in the TRH-induced increase in the Indo-1 fluorescence ratio. Figure 6 presents grouped data from 7 replicate
60i
S
50-
50-
40-
30,,11111
FlG. 4. Effect of TRH on Indo-1 ratios in AP cells in the presence of 2 mM EGTA. The left panel is a three parameter scattergram showing a representative TRH-induced change in Indo-1 ratio of AP cells in medium with normal Ca2+. Addition of 2 mM EGTA at time channel 2 causes a gradual decrease in the ratios, which stabilize by time channel 16. Addition of 1 nM TRH at time channel 23 causes a transient increase in a subpopulation of AP cells. The right panel shows a similar experiment in AP cells that were pretreated with 100 nM sCT for 5 min prior to addition of TRH. sCT almost totally blocks the effect of TRH.
20
Time (seconds x 4.7)
Time (seconds x 4.7)
2
20- I
20•
10-
10-
10
20
30
40
50
Time (seconds x 4.7)
§i™l I l l l IIIHIIfJlfllMIII III Illllllllllh 10
20
30
40
50
Time (seconds x 4.7)
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 November 2015. at 00:30 For personal use only. No other uses without permission. . All rights reserved.
60
CT INHIBITS PITUITARY Ca2+
617
1000 200 •
X•2
^V
g 600 n
>
^
20
30
40
S S
15--
200 >•• • i
0
10
20
20
30
Time Channel (seconds x 4.7) FIG. 5. Effects of EGTA on the number of cells responding to TRH. The plot shows responses to 1 nM TRH in the presence and absence of 2 mM EGTA from three replicate experiments in each group. The mean ± SEM number of cells exhibiting fluorescence above threshold in the absence of EGTA ( • D) and in presence of EGTA ( • • ) , which was added at the start of the scan, are depicted. The inset highlights the difference in the groups during latter part of the responses (secondary phase). Note that the standard error bars are frequently smaller than the mean symbols. 28 A
«
26--
CO
5 o S
TRH
24-
ij
22
j
40
50
60
FlG. 7. Effect of sCT on TRH-induced increases in Indo-1 ratios in EGTA-treated AP cells. The plot presents the overall population mean ± SEM Indo-1 ratio data from 7 replicate 300-sec scans in each condition. Both groups of cells were exposed to 2 mM EGTA at time channel 9 and then to 1 nM TRH (=) at time channel 21. Control cells (A A) show a transient increase in Indo-1 ratio in response to TRH, while a 5-min pretreatment with 100 nM sCT ( ,• • ) significantly inhibited the overall Indo-1 ratio response (P < 0.001, unpaired t-tests on digitized areas under the curves).
to 3-5% (P < 0.001, t-test). sCT also significantly curtailed the brief initial Indo1 ratio response to TRH in cells treated with 2 mM EGTA, as shown in the right panel of Fig 4 as a representative individual example, and in Fig 7, depicting group means ± SEM from 7 scans per treatment condition. In these studies, the mean Indo-1 ratios were reduced by the sCT treatment, and then further reduced by EGTA as expected, prior to the addition of TRH.
TJTTTT
Discussion
20
