NASA Technical Reports Server (NTRS) 19970025924: Experimental Modification of Rat Pituitary Growth Hormone Cell Function During and After Spaceflight

16 Pages·1.3 MB·English

by  NASA Technical Reports Server (NTRS)

c3.3 '70
NASA-CR-205106
Experimental modification of rat pituitary growth
hormone cell function during and after spaceflight
W. C. HYMER, R. E. GRINDELAND, T. SALADA, P. NYE, E. J. GROSSMAN, AND R K. LANE
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University
Park, Pennsylvania 16802; and National Aeronautics and Space Administration Ames Research
Center and Lockheed Engineering and Sciences Company, Moffett Field, California 94035
Hymer, W.C., R. E. Grindeland, T. Salada, P. Nye, E. J. ingly, many of these changes persisted for 2 wk post-
Grossman, and P. K. Lane. Experimental modification of flight (15, 16).
rat pituitary growth hormone cell function dm'ing and after This report describes changes in rat GH cells them-
spaceflight. J. Appl. Physiol. 80(3): 955-970, 1996.--Space- selves during and after an 8-day spaceflight with a
flown rats show a number of flight-induced changes in the
passive cell culture system. The most important changes
structure and function of pituitary growth hormone (GH}
found were those relating to 1) the amount and biologi-
cells after in vitro postflight testing (W. C. Hymer, R. E.
cal activity of GH released from cells in vitro, 2) the
Grindeland, I. Krasnov, I. Victorov, K. Motter, P. Mukherjee,
responsiveness of the GH cells to hydrocortisone (HC)
K. Shellenberger, and M. Vasques. J. Appl. Physiol. 73,
and hypothalamic GH-releasing hormone (GHRH), 31
Suppl.: 151S-157S, 1992}. To evaluate the possible effects of
the cytoarchitecture of GH cells, and 4) their intracellu-
microgravity on growth hormone (GH) cells themselves,
freshly dispersed rat anterior pituitary gland cells were lar hormone content. Evidence for recovery of some, but
seeded into vials containing serum _+1 pM hydrocortisone not all, of these changes during a 6-day postflight test
(HC) before flight. Five different cell preparations were used: period was obtained. Finally, the data show the influ-
the entire mixed-cell population of various hormone-produc- ence of paracrine interactions between the heteroge-
ing cell types, cells ofdensity < 1.071 g/cm a(band 1), cells of neous cell types on cell function as they experience low
density > 1.071 g/cma Iband 2), and cells prepared from gravity.
either the dorsal or ventral part of the gland. Relative to A companion report describes changes in PRL cells
ground control samples, bioactive GH released from dense that also occurred during this same experiment (18).
cells during flight was reduced in HC-free medium but was
MATERIALS AND METHODS
increased in HC-containing medium. Band 1and mixed cells
usually showed opposite HC-dependent responses. Release of Animals and Tissue Processing Before Flight
bioactive GH from ventral flight cells was lower; postflight
Animal care and use for this experiment, which was flown
responses to GH-releasing hormone challenge were reduced,
on the Space Shuttle in 1992 (STS 46), were approved by
and the cytoplasmic area occupied by GH in the dense cells Institutional Animal Care and Use Committees at both The
was greater. Collectively, the data show that the chemistry
Pennsylvania State University and the National Aeronautics
and cellular makeup of the culture system modifies the and Space Administration (NASAl Ames Research Center
response of GH cells to microgravity. As such, these cells offer and conformed to National Institutes of Health guidelines.
a system to identify gravisensing mechanisms in secretory Nineteen hours before launch, 100 specific pathogen-free
cells in future microgravity research. Sprague-Dawley male rats (200-220 g; Harlan Sprague
microgravity; cell culture; cell gravisensing; growth hormone Dawley, Frederick, MD) were killed by decapitation and their
entire anterior pituitary glands or the dorsal and ventral
assays
regions were dissociated into single-cell suspensions with a
trypsinization technique that routinely yields 2-2.5 × 10_
cells from each gland (17).
Cells 12.0 × 10_ in 200 _1) from one of five different
IF AND HOW CELLS SENSE the low gravity of spaceflight
experimental groups {Fig. 1)were added to a 4.4-ml capacity
has been thoughtfully considered, modeled, and de-
borosilicate glass vial (Wheaton_ containing 4.0 ml of culture
bated for years. Many different types of cells have been medium [either modified Eagle's minimal essential medium
exposed to microgravity (14); some show significant and (_MEM) containing 5_ calf serum, 0.2% NaHCO:_, 25 mM
repeatable changes (4-6, 19, 24). Although this area of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)
investigation is only beginning, many believe that buffer (pH 7.4), and antibiotics; or _MEM containing insulin,
transferrin, and selenium (Collaborative Biomedical) buff-
these kinds of studies will 1) eventually help to define
ered with 25 mM HEPES (pH 7.4) and antibiotics)} 128, 33).
the mechanisms that cells and organisms use to re-
Some of these media were supplemented with 1I_M HC. The
spond to this unique environment and 2) help to cell groups included 1) the entire population of different
explain the well-documented changes in the musculo- hormone-producing cell types (mixed), 21 separated cells of
skeletal, immune, vascular, and endocrine systems of low density [1.040-1.071 g/cma; (band 1)],31 separated cells
spacefiown animals and astronauts (11, 12). We studied of high density [1.071-1.085 g/cm37, (band 2)], 4) mixed cells
growth hormone (GH) cell structure and function in prepared from the dorsal part ofthe gland, and 5) mixed cells
three previous spaceflight experiments because pitu- from the ventral part of the gland. A dorsal and ventral
section from each gland was made with a Smith-Farquhar
itary GH participates in the regulation of these organ
tissue chopper. Each gland was positioned on the stage of the
systems. We found that significant changes had oc-
device so that a single slice would reproducibly yield two
cuffed in the GH and prolactin (PRL) cells prepared sections of _6,000 _m each. The method used to separate GH
from animals in microgravity for 7-14 days; interest- cells into two subpopulations involved layering 107dispersed
955
956 GROWTH HORMONE AND SPACEFLIGHT
85 pituitary 15 pituitary change on day 3. Cells in other vials from mixed, band 1, and
glands band 2 flight and ground groups (only those in serum-
containing medium without HC) were tested for their respon-
cells dorsal section ventral section siveness to a synthetic hypothalamic GHRH (2 × 10 _ M;
Peninsula Laboratories). This testing was done by 13 succes-
cells cells sive additions and replacements of fresh medium (1 ml of
mixed s_ uMEM + 5% calf serum each time) to each cell-containing
vial (n = 3 vials/treatment group) at 15-min intervals. Only
cells cells the fourth and ninth medium changes contained either 2 ×
i ligh4t, hea4v,y 10 -9M GHRH prepared in phosphate-buffered saline (1 pl) or
¥
vials vials vials vials vials vehicle alone.
GH Assays
S
Concentrations ofimmunoreactive GH (iGH) released from
the cells into the culture media were determined by an
flight ground ground
(19 hours) enzyme immunoassay (8). The polyclonal antiserum to GH
(8 days) (8djays) has a cross-reactivity of <0.3% to PRL at the final dilution
(1:80,000) used in the assay; each sample was analyzed in
post flight processing I process at launch duplicate at two dilutions, and the results are expressed
released GH released GH relative to a rat GH standard preparation (B-11) kindly
GHRH testing intracellular GH provided by the National Institute of Diabetes and Digestive
intracellular GH and Kidney Diseases (Bethesda, MD) and the National
morphology Hormone and Pituitary Program (University of Maryland
School of Medicine, Baltimore). Intracellular GH was ex-
Fig. 1. Experimental design. GH, growth hormone; GHRH, GH- tracted from the cells by overnight incubation in 0.01 N
releasing hormone. See MATERIALSANDMETHODSfor details. NaHCO3 (4°C), followed by centrifugation (1,000g for 30 min)
to remove particulate material. Under these conditions, the
cells (1 ml) over discontinuous density gradients of bovine supernatant fraction contains >90% of the extractable GH
serum albumin (29). Layer 1 had a density of 1.071 g/cm _, (9).
whereas layer 2 had a density of 1.085 g/cm 3. After cen- Concentrations of biologically active GH (bGH) in the
trifugation (2,000 rpm for 30 min at 4°C), cells were col- culture media and extracts were determined exactly accord-
lected from the two layering interfaces. Those of dens- ing to the tibial-line bioassay procedure of Greenspan et al.
ities < 1.071 g/cm 3(band 1) have GH cells with relatively few (10). Approximately 2,000 hypophysectomized female rats, 26
cytoplasmic GH-containing 0.3-_am secretory granules, days old at surgery, were used to assay samples with a
whereas those of densities > 1.071 g/cm 3(band 2) are laden four-point assay procedure (i.e., four rats/dose at two doses).
with these particles (29). The average recovery of cells from The assay's end point measures increases in tibial epiphyseal
the gradient was 86% in = 7 preflight trials), and the GH cell plate widths after four daily injections of hormone; it has a
percentages [determined by flow cytometry (15)] in nine trials sensitivity of 1 _g and is specific for GH. Responses were
were 23 _+ 3 and 52 _+ 4% for band I and band 2 cells,
compared with a bovine GH standard (1.5 U/mg) calibrated
respectively. Other preliminary data indicated that band 2 against a USP standard; they are expressed in terms of an
GH cells contained 100 pg GH/cell, whereas band 1 cells in-house preparation of rat GH (3.0 IU/mg).
contained about one-half of that amount. The earlier report of
Snyder et al. (29) showed that the addition of 1 _M HC to High-Performance Liquid Chromatography (HPLC)
serum-containing medium markedly increased the intracellu-
lar GH of cultured pituitary cells but had relatively little To compare apparent molecular weights of GH released
effect on hormone release; preliminary experiments with 1 from cells in microgravity vs. unit gravity, 1-ml samples of
_M HC in the closed-vial system confirmed this result. serum-containing media (÷ 1 _M HC) from the mixed, band 1,
There were six sealed cell-containing vials for each of the and band 2 flight samples in = 3 samples/group) or their
five experimental groups; the total number of vials exposed to corresponding ground control samples were lyophilized and
microgravity was 165. Three identical sets of 165 cell- reconstituted in 500 _l of 0.1 M potassium phosphate buffer
containing vials were kept at 37°C between the time of containing 0.05 M NaC1, pH 7.8. Each sample was applied to
preparation (launch minus 19 h) until 12 h before landing a sizing column of Protein-Pak 300 SW (7.8 mm × 300 mm;
when they were required to be kept at 22°C. One of the 165 Waters, Milford, MA) equilibrated with the same buffer. The
vial sets served as the synchronous ground control; it was column flow rate was 0.3 ml/min, and 0.6-ml fractions were
collected. The column was calibrated with blue dextran (mol
kept in the laboratory at the Kennedy Space Center (FL)
under conditions identical to those in space during the 8-day wt 2,000,000), [3-amylase (mol wt 200,000), bovine serum
flight. The other vial set was processed at launch to determine albumin (mol wt 66,000), carbonic anhydrase (mol wt 29,000),
how much GH was released from the cells into the medium and ribonuclease (mol wt 13,683).
during the 19 h between the end of cell preparation and the
Morphology
launch.
In some cases, cells were removed from the vials by
Postflight Cell Culture and GHRH Testing
trypsinization (19) and prepared for immunocytochemistry or
Postflight processing began after transport to the testing flow cytometry. Immunocytochemistry was used to identify
facility (7 h). Some of the cell-containing vials (n = 3 GH cells in preparations that were attached to poly-a-lysine-
vials/flight and 3 vials/ground) were replaced with fresh coated coverslips (15). Briefly, this involved fixation in Zambo-
media, and postflight culture continued in the same sealed ni's fluid followed by membrane permeabilization with 0.4%
vials for an additional 6 days with an intervening medium Triton X-100, incubation in GH antiserum (1:10,000) for 36 h,
GROWTHHORMONAENDSPACEFLIGHT 957
incubatioinnhorseradispheroxidasaentiserum(1:500a),nd
Closed-Cell Culture System: Validation
stainingwithdiaminobenzediTnhee.sepreparationwsere
usedfordigitalanalysiosfthecytoplasmaicreaoccupiebdy Because it was not possible to do this experiment on the
GH.Thisproceduraendequipmenwtereidenticatlothose Space Shuttle with conventional cell culture hardware, a
usedpreviousl(y16);it generateasdigitizedimageofthe simple passive system was developed. Release of iGH from
stainedcell,colorizedaccordintgointensitieswithinthe cells in the closed-vial system over a 9-day period at 37°C was
imagedevice2's56graylevelstoquantitatetheareaofGH linearly related (r2 = 0.992) to cell number over the range of
stainingA.sbeforec,arewastakentomaintainconstanlitght 103 to 8 x 10s cells seeded (9 trials). The amount of iGH
settingsc,ondensheeright,andaperturesizeinadditionto synthesized by 2 x 10s cells during the 9 days (i.e., the
normalizinvgariationsinlightwithalightmeterplacedat amount recovered in media plus cells minus the amount
thelevelofthecameraI.n somecasest,herewerealso initially seeded) ranged between 15 and 20 1Jg. A majority of
sufficienctellstodoflowcytometriacnalysisofGH-stainedsynthesized GH was released into the culture medium. Net
cellsin suspensio(n15).Cellfixationwasin phosphate-GH synthesis in the closed-vial system was not significantly
bufferesdaline-azide-buffe4r%eFdormalinfollowebdymem- different from that measured in primary rat pituitary cell
branepermeabilizatiwointh0.4%TritonX-100in,cubatioinn cultures that are routinely maintained on conventional plas-
GHantiserum(1:10,00o0v)ernighitn,cubatioinnfluoresceinticware (Linbro tissue culture plates) that allows free gas
isothiocyanate-conjugaantetisderumc,ounterstaininwgith exchange (95c_ ,air-5% CO2) (33). It was also not significantly
different from that measured in cells cultured on Linbro
propidiumiodide,andanalysisby flowcytometry(Epics
mode7l 53).Parameteresvaluatedon30,000cells/sampleplates that were sealed to prevent gas exchange (data not
shown).
were
1)GH cell percentage, 2) marker index (the ratio of the Several formulations of culture media were tested to
voltages of stained to unstained cells; an index of the "bright- determine optimal GH synthesis in the closed system; these
ness" of fluorescence staining), 3) forward-angle light scatter included Medium 199, aMEM, addition of 0.2% NaHCO3, and
(FALS; an indicator of cell size), and 4) perpendicular light calf or horse serum (either 5 or 10%). In addition, we
scatter (PLS; an indicator of the content of cytoplasmic compared GH synthesis levels in cells cultured in rat serum
hormone-containing secretory granules) (15). The application with those cultured in calf or horse serum. The rank order
of the flow cytometer to evaluate these parameters for rat was determined to be rat serum > calf serum > horse serum.
pituitary cells, as well as the experimental data that establish Cells in rat serum synthesized 2.3 _+ 0.4 times as much
their biological significance, has been documented (25). hormone as those in calf serum (n = 14 experiments);
Fig. 2. Electron micrograph of a GH and prolactin cell maintained in a closed glass vial for 9days at 37°C before
trypsinization and preparation for electron microscopy. Cell ultrastructure compares favorably with cells
maintained under more usual culture conditions. Magnification, x25,000.
958 GROWTH HORMONE AND SPACEFLIGHT
Initial Ground Flight
Mixed
.,.,?.
.".:1._t!
>:,i.:t; I ' !
,:,:, , ?:.£-:-':"1. '.i:::
-./
t, _ L
!i,_'_;- ' .
!#'!i. iii.
Fig. 3. Flow cytometric histograms of
pituitary cells contained in the mixed ,-_ I-'l'i'ii]i|i': ' t'_hlll , _,l_ ltilii ,i Iliil
(top row), band 1 (middle row), and LF"3FL 1 LPBFL I oEn_ 1 LPGFL I000
band 2 (bottom row) samples before
seeding into glass vials (initial; left
column) and after 9 days of culture
under unit gravity (ground; middle col- Band 1
umn )or microgravity (flight; right col- _=
umn) conditions. Log peak green fluo-
rescence (LPGFL) staining is specific
for GH cells and delineates stained
_r I ;_- • •
[identified as cells falling to right of
vertical gate (vertical line) in each _.
panel] from unstained cells that in-
elude other hormone-producing cell : ;_"_' -::':. _ i
types gated from debris on basis of
propidium iodide nuclear stain. LPGFL .,. i i iHHI i Ilii*ll _IIII ,m :: Ill :i ..IIill.lll I_--_l " " '
is on a log scale, whereas forward-angle I00( I lO00 1 IOOF_
LPC--FL !..PGFL LP_3FL
light scatter IFALS}, a measure of cell
size, is linear. Each pattern represents
a count of 30,000 cells.
Band 2
' . t _il i '.':' .i.... : ,_'_/ . t
" " o_1..
_c,:, i_i#6' I ........ _._'_
however, it was not selected for use in the flight experiment occur during trypsinization. Amounts of GH released from
because rat serum supplies were limiting. With 2 > 10_cells every 1,000 GH cells seeded during 9 days in culture was
in closed vials for 9 days, the pH is maintained within 0.1 unit 336 _+9 ng (n = 15 experiments).
of its initial value (pH 7.4).
After 9 days, the cells showed a typical ultrastructure Mierogravity Cell Culture: Special Considerations
including intact membranes around the secretion granules
(Fig. 2). Other data showed that 53 -+ 3% (n = 14 trials) of the It was necessary to establish that the vibration forces
cells originally seeded could be recovered by trypsinization associated with the Shuttle launch and recovery would have
after 9 days; the percentage of recovered GH cells (29 -+ 3%; no deleterious effect on cell attachment, structure, or func-
n = 11 experiments) compared favorably with input percent- tion. We found >97% of the cells seeded into the vial routinely
age, demonstrating that preferential loss of GH cells did not attached to the vial bottom within 18 h; furthermore, they
Table 1. Effect of microgravity on cell parameters measured by flow cytometry
GH Cells, q Total Cell Population GH Cell Population
After flight FALS PLS Marker index FALS PLS
Before
flight Flight Ground Flight Ground Flight Ground Flight Ground Flight Ground Flight Ground
Mixed 32 12 11 21 25 21 33 5.2 6.1 23 24 57 66
Band 1 22 9 11 27 30 29 37 4.4 3.9 20 23 34 49
Band 2 57 47 62 23 27 42 58 2.1 2.2 25 29 75 84
Values represent peak channels of size [forward-angle light scatter (FALS)] and cytoplasmic granularity [perpendicular light scatter (PLS)]
and ratio of voltages of stained to unstained cells (marker index)• Data were collected from 30,000 cells/sample. GH, growth hormone.
GROWTH HORMONE AND SPACEFLIGHT 959
remained attached after vibration tests were conducted at the GH released over the entire 8-day culture period; these data
NASA Ames Research Center that were greater than those are therefore not included in RESULTS.
typically experienced on the Shuttle. Furthermore, shaking Approximately one-half of the cell culture vials used a
had no effect on subsequent release of iGH during a 9-day chemically defined serum-free medium that was supple-
culture test. mented with insulin, transferrin, and selenium. This formula-
Because there was a 0.2-ml air bubble at the top of each vial tion was used to eliminate possible effects of unknown serum
and because bubble behavior can be unpredictable in space, factors on GH cells. In every case, the trends in GH release
we were concerned that it could become dislodged in flight, between various treatment groups were identical to those
come to rest directly over the cell layer, and result in cell using serum-containing media except that the actual GH
dehydration. To discourage this possibility, each vial was levels were consistently lower. We have therefore chosen not
rimmed internally (upper one-third) with a surface active to present these results in this report.
agent (Prosil 28, Thomas Scientific); furthermore, one time Two automatic temperature recording devices, developed
each day, an astronaut rotated the vial container five times in at the NASA Ames Research Center, were secured in the
a period of 10 s. sealed vial containers to continuously monitor ambient tem-
We also conducted a trial aboard the KC-135 airplane to perature around the vial sets during spaceflight and in the
study bubble behavior in the glass vial during the 20-30 s of laboratory at the Kennedy Space Center. The sensitivity of
microgravity achieved in parabolic flight and verified that the the temperature recorder was _+0.4°C. The recordings indi-
air bubble tended to remain in the upper one-third of the filled cated that the control cells experienced temperatures ranging
vial.
between 37.3 and 37.6°C for the 9-day experiment while the
Data Presentation flight cells experienced temperatures between 38.4 and 38.8°C
for the same period. Further analysis indicated that the
We determined that the amount of GH released during the average temperature difference was 1.2°C between flight and
19 h before the Shuttle launch (Fig. 1) was <1% of the total ground. The entire flight experiment was therefore repeated
A
B
C D
Fig. 4. Examples ofGH cells stained immu-
nocytochemically {A, C, and E) and their
digitized color images (B, D, and F) used to
quantitate their cytoplasmic areas occupied
by GH. Examples of GH cells in mixed (A
and B}, band 1 tC and D), and band 2
(E and F) cells are shown. Magnification,
×1,500.
E F
960 GROWTH HORMONE AND SPACEFLIGHT
Immunoassay
Extracellular Intracellular
Mixe._ Mixed
300" 60
Flight Postflight Flight Postfiight
200" + Ground
X 40
---[3-- Flight
without hydrocortisone
100' 20'
0
8 10 12 1_, '
300- Band1 60 Band1
¢n
Fig. 5. Release (extracellular) and content (intra-
cellular) ofimmunoreactive GH (iGH) from mixed 3=
(top), band 1(middle), and band 2 (bottom) cell CD 200 40
samples without hydrocortisone during and after
microgravity. Data are means _+SE expressed o
relative to number of GH cells seeded into vials;
n = 3 vials. Error bars not shown fit within 100 20
symbol. *P < 0.05.
c-
0 i i i i
12 14 8 10 12 14
300" Band2 60
200' 4O
100" 20'
t3
• i ] l • i , / ' i
8 10 12 14 8 10 12 14
Days in Culture
6 mo later in the laboratory at The Pennsylvania State vial. The general morphology of the cells after space-
University where cells in the closed vials were kept at either flight, while still attached, was unremarkable and
39 (to mimic flight temperatures) or 37°C for 9 days. A similar to the cultured ground control samples; steroid
statistical comparison of the data between these two experi- treatment had no obvious effect on cell morphology.
ments with a logarithmic transformation methodology fur-
Flow cytometric histograms of GH-specific log peak
ther showed that the changes in the flight experiment could
green fluorescence vs. FALS signals show that several
not be explained on the basis of a temperature differential.
We have therefore chosen to present only the synchronous changes had occurred between the time of initial cell
ground control data in this report. preparation and recovery from the vials after the
Experimental constraints within this spaceflight investiga- 10-day culture. These included 1) broader FALS pat-
tion resulted in low n sizes. Accordingly, multiple s-level- terns in unstained cells (mixed and band 1 samples)
corrected independent t-tests were used to analyze these after culture than before culture, 2) decreased FALS
data. This, in turn, permitted use of an inferential technique
signals of unstained cells in band 2 during the ground
to highlight differences as well as strong trends. Significance
was maintained at P <- 0.05. and flight cell culture periods, and 3) maintenance of
the FALS pattern of GH cells in the band 2 sample
RESULTS during the entire experiment but 4) a lack of mainte-
nance of the FALS pattern of GH cells in the mixed and
GH Cell Morphology
band 1 samples over the 10-day period (Fig. 3). Table 1
The viability of all cell samples both before and after shows data derived from these histograms that com-
flight was >90c_ :.At the end of the experiment, the pH pare the effects of flight on 1)the GH cell frequency, 2)
of the culture medium was 7.4. Microscopy showed that the mean FALS-to-PLS peak channel of the total and
all cells had remained attached to the bottom of each GH cell populations, and 3 )the marker index of the GH
GROWTH HORMONE AND SPACEFLIGHT 961
Immunoassay
Extracellular Intracellular
Mixed Mixed
Postflight
300] Fhght*<, Postflight 60 Flight ,'
+ Ground
200 40 ,
---o-- Flight
with hydrocortisone
10:
300- Band 1 60 _
59
Fig. 6. Release and content of iGH from mixed
O
"1- (top), band I (middle), and band 2(bottom) cell
(.9200- 4O samples maintained in hydrocortisone-contain-
O ing medium during and after microgravity. Data
OO are means _+SE expressed relative to number of
GHcells seeded into vials; n 3vials. Error bars
"" 100-
"1- not shown fit within symbol. *P < 0.05. *":P <
o_ v 0.01.
u---'---'--" f "
r.-
0 i
i i i i
8 10 12 14 :' 1_0 1'2 1'4
300- Band2 6o
i
200" 40
100" 2O
• 8' ' 10" ' 1'2 ' 1'4 ' t0 12 14
i I ' ] ' T ,
Days inCulture
cells. Because PLS signals are on a logarithmic scale areas that contained hormone. This was not true in
(not shown in Fig. 3), the lower mean channel values of band I cells. GH percent area occupancies measured on
all flight cells are considered significant. On the other 200 cells/sample were 40 _+3% for ground vs. 53 +_2%
hand, differences in the FALS and marker index were for flight mixed samples (P < 0.001), 39 -+ 3c_, for
small (linear scale); we conclude that neither the ground vs. 37 -+ 2% for flight band 1 samples (not
fluorescence intensity of GH staining nor the size of the significant), and 27 + 2% for ground vs. 40 _+ 2c_,for
pituitary cells was affected by spaceflight. Determina- flight band 2 samples (P < 0.001). Examples ofimmuno-
tion of GH cell frequency by manual counting (not cytochemically stained GH cells and their digitized
shown) was consistent with the data in Table 1. We images used to collect these data are shown in Fig. 4.
attribute the changes in GH cell FALS patterns after
the experiment to the cell culture procedure per se; Extracellular and Intracellular GH
supporting evidence for this idea can be found in Ref. During and After Flight
15. However, we have no explanation why band 2 cells
maintain discrete FALS patterns when they are in iGH +- HC. Relative to ground control samples,
isolation but not when they are present as a part of the microgravity had no effect on the release of iGH during
mixed sample. the 8 days in space nor during the subsequent 6-day
Image analysis of individual GH cells from both postflight culture period. Immediately on landing, the
mixed and band 2 groups after flight indicated that, intracellular contents of iGH were significantly lower
relative to their ground control samples, there were than in the ground control samples; however, this
statistically significant increases in their cytoplasmic difference was not maintained after the postflight
962 GROWTHHORMONAENDSPACEFLIGHT
Bioassay
Extracellular Intracellular
Mixed Mixed
2000 600-
Flight Postflight Flight ' Postflight
i
I
1500 i
400- i
1000
200"
5OO
0
I I i i i , i i
10 12 14 8 10 12 14
CD 2000 Band1 6007
Fig7..ReleaasnedconteonftbioactiGveH(bGH) O
frommixed(top)b,and 1(middle), and band 2 1500
(bottom) cellsamples without hydrocortisone dur- 400"
0
ingand after microgravity. Data are means ± SE 0
0 1000
expressed relative to number ofGH cells seeded
into vials; n = 3vials. Error bars not shown fit
within symbol. _*P < 0.01.***P < 0.001. 500-
.Q
D_,,,____ 200"
C
0
i i i f
10 12 14 8 10 12 14
Band2 + Ground
2000 ]Band2 600- ** "-_-- Flight
:!1i! o
400'
_'_thouthydrocortisone
' i . i , I . i , i
8 10 12 14 8 10 12 14
Days inCulture
culture (Fig. 5). The addition of HC to the culture the presence of HC than in its absence; this was not
medium marginally increased iGH release from the true for GH cells in the band 2 samples. Effects of
mixed ground cell sample and caused a twofold in- microgravity on intracellular GH were complex• In
crease in the intracellular iGH in band 2 cells of both general, flight cells cultured without HC often con-
ground and flight groups; the relative difference in tained significantly less bGH, but all cell groups cul-
intracellular iGH between these groups measured in tured in the presence of HC in microgravity consis-
the absence of HC (Fig. 5) tended to continue in the tently contained significantly less bGH.
presence of HC (mixed, P < 0.055; band 2, P < 0.057; HPLC analysis of extracellular iGH. Culture media
Fig. 6). taken from cells after 8 days in microgravity were
bGH +_HC. Relative to the ground control samples, found to contain iGH molecules of widely different
major changes in the release of bGH, especially in apparent molecular weights. In general, neither micro-
HC-containing media, were found (cf. Figs. 7vs. 8). In gravity nor steroid had any marked effect on the size
Earth's gravity, more bGH was released from band 2 distribution profile of the hormone (Fig. 9). The most
cells than its flight counterpart; addition of HC re- important change was the tendency for flight samples
versed the effect. In space, the opposite was true; i.e., to contain more GH of higher apparent molecular
HC stimulated bGH release from band 2 cells back to weight. To make statistical comparisons between the
the levels of non-HC-treated earthbound band 2 cells. amounts of iGH contained in different molecular-
This response continued into the postflight period. weight regions of these chromatograms as a function of
Band 1 cells from both ground and flight samples gravity level, cell sample, and steroid, we compared the
released considerably more (two to three times) bGH in sums of iGH contained in three different molecular-
GROWTHHORMONAENDSPACEFLIGHT 963
Bioassay
Extracellular Intracellular
Mixed Mixed
2000- 600-
Flight Postflight Flight Postflight
1500-
1000-
400' ,_
500-
200'
i
I I
8I t' 10I 12I 14I i8 t, 1() 12i 1_,
Bancll ' Band1 :
2000
Ground
o ----43-- Flight Fig. 8. Release and content of bGH from mixed
©I 1500 with hydrocortisone (top), band 1(middle), and band 2 {bottom)cell
O [_ I 400 *** samples maintained in hydrocortisone-contain-
O ing medium during and after microgravity. Data
2 1000
600 are means _+SE expressed relative to number of
I GHcells seeded into vials; n 3vials. Error bars
500 2OO not shown fitwithin symbol. **P < 0.01. ***P <
0.001.
C
0 15
10 1 14
Band 2 Band2
2000--- 600-
1500-
400'
1000
500
i _ i , i , 1 i _ i i i
8 10 12 14 8 10 12 14
Days inCulture
weight regions (high, 9.9 x 105 to 2.4 x 105; medium, were only moderate to nonexistent, whereas the re-
2.4 x 105 to 5.9 x 104; low, 5.9 x 104 to 1 x 104). The sponses of ground-based mixed and band 1 cells were
only statistically significant effect (P -< 0.05) was the significant (Fig. 10). The lower sensitivity of the GH
reduced amount of iGH recovered from the low- bioassay required combining five 15-min fractions (in-
molecular-weight regions of the mixed HC-treated cell cluding the GHRH pulse) before assay. These results
flight samples (Fig. 9D). Strong tendencies toward also showed 1) nonresponsiveness of mixed cells after
differences between flight and ground were noted in the flight (Fig. 11), 2) apparent increased sensitivity of
following samples: 1) mixed, without HC, high molecu- band 1 cells to GHRH (Fig. 12), and 3) weak to
lar weight: flight > ground (P < 0.08); 2) band 1, with nonexistent responses of band 2 cells (Fig. 13).
HC, high molecular weight: flight > ground (P < Substrate. Three vials pretreated with Matrigel (a
0.07); and 3) band 2, without HC, medium molecular commercially available mixture of basement mem-
weight: flight > ground (P < 0.10). In general, the total brane macromolecules) and then loaded with mixed
amounts of iGH recovered after HPLC from flight and cells were also exposed to low gravity. This treatment
ground samples were within 10% of each other. Rela- had no effect on the release of either iGH or bGH from
tive to separated cells, a greater fraction ofiGH was of cells regardless of treatment group, i.e., flight, ground,
higher apparent molecular weight when the total cell and postflight (data not shown). Band 1 and 2 cells
population was used. The reason for this difference is were not tested.
not known. Cell location. Flight had no effect on the release of
Response to GHRH. After 8 days in low gravity, cell iGH from mixed cells prepared from either dorsal or
responses (iGH release) to 10 9 M GHRH challenge ventral regions of the pituitary gland [dorsal, 367 _+22
964 GROWTH HORMONE AND SPACEFLIGHT
0 0
0 0 00
1500 Mixed cells Mixed cells T ---e-- Ground
---o-- Flight
lOO0 (- HC)
500-
(+ HC) _
--EI.-O-.EI-E]-O-O-O"_ . , T -%]"(3
0 --0--0--{3--0-[:}--0-'(:3-[
o 5 10 15 20 5 10 15 20
15oo- Band 1cells Band 1cells
t-
O
O
1000"
(- FIC) (+ HC) ._
1-
500'
t-"
0
0 5 10 15 20 0 5 10 15 20
15oo Band 2 cells Band 2cells
looo (- HC) (+HC)
6 =_=o 1_'I'KTk
o 5 10 15 20 5 10 15 20
Fraction Fraction
Fig. 9. Fractionation of culture media without (-HC) and with hydrocortisone (+ HC) from spaceflown and ground
control cells by gel exclusion high-performance liquid chromatography In 3 separate sample media/group).
Elution position of standard molecular-weight markers (see MATERIALS ANDMETHODS) are shown in upper left panel.
For ease of comparison of these data, see Table 2.
(flight) and 347 _+ 14 ng/1,000 GH cells (ground); tuated that difference (Fig. 14C). However, a similar
ventral, 150 _ 7 (flight) and 169 _+7 ng/1,000 GH cells analysis of iGH from band 2 flight cells give very
(ground)]. However, release of bGH from ventral flight different profiles; in this case, HC reversed this pattern
cells was reduced by one-half [dorsal, 116 _+4 (flight) (Fig. 14D). Not surprisingly, the mixed cell sample gave
and 108 +_ 15 ng/1,000 GH cells (ground); ventral, a pattern that was different (Fig. 14B). Comparison of
240 _+ 10 (flight) and 545 _ 19 ng/1,000 GH cells the molecular-weight data, i.e., patterns of iGH vari-
(ground); P < 0.001]. ants contained inside cells (Fig. 14) vs. those released
Intracellular GH variants. Western blots of cell ex- from cells (Fig. 9), show an obvious correlation between
tracts after separation by sodium dodecyl sulfate- the two data sets. Heterogeneity of molecular forms of
polyacrylamide gel electrophoresis under nonreducing GH are known to result from mRNA splicing, posttrans-
conditions revealed a complex array of iGH forms that lational modifications (e.g., aggregation or glycosyla-
were qualitatively similar between flight and ground tion), and proteolytic cleavage.
samples (Fig. 14A). Analysis of these various cell
samples by densitometry showed that although differ- DISCUSSION
ences between flight and ground did not vary within
any given region by >10%, the pattern of changes The primary objective of this cell culture experiment
depended on both the sample and presence of HC. For was to find out whether the postflight changes in
example, flight tended to increase amounts of higher pituitary GH cells prepared from rats after 7-14 days
molecular-weight iGH forms in band 1 cells at the in space also happened when GH cells themselves were
expense of lower molecular-weight forms and HC accen- put into space. Within the constraints of the hardware