FINAL REPORT ON GROWTH HORMONE RELEASING

FACTOR (GRF) BINDING SITES OF PITUITARIES

OBTAINED FROM SPACE FLIGHT RATS.

A.K. Malik and R.E. Grindeland

(Dept. of Biology, San Jose State University, CA, Life Sciences Division

NASA Ames Research Center CA)

[Abstract]{.underline}

Growth hormone secretion is compromised during exposure to actual or
simulated (rat hindlimb suspension) microgravity. Further, growth
hormone secretion in response to growth hormone releasing factor (GRF)
are markedly reduced in pituitaries of suspended rats. These data
suggest that GRF receptors may be reduced by microgravity, in number of
binding affinity, thus, this study was designed to understand whether or
not, the growth hormone releasing factor (GRF) binding sites are altered
in terms of their number (binding capacity) or affinity upon exposure to
microgravity. Radiolabelled GRF (human) was used as a radiogland and
with rat GRF as a cold competitor the receptor assay was performed on
the pituitary homogenates prepared from the male albino rats, flown on
the SLS-1 Mission. We did not find any specific (receptor) binding
presumably due to the severe desiccation of the glands. In contrast,
homogenates prepared from the freshly dissected glands, using the same
procedure, provided a dose-response curve.

[Background]{.underline}

Growth is a process regulated mainly by growth hormone (GH) secreted by
the anterior pituitary cells called somatotrophs. The release of growth
hormone is directly under the control of two hypothalamic peptides: the
stimulatory growth hormone releasing factor (GRF), and an inhibitor
somatostatin (SS) (11). GRF is primarily secreted by the arcuate nucleus
of the hypothalamus that directly projects to the external layer of the
median eminence near capillary loops forming portal vessels to the
pituitary gland (1,2,3,4). Other cell bodies secreting GRF are detected
in the dorsomedial, ventromedial, and paraventricular nuclei, as well as
in the fornical regions (12). GRF acts on the somatotrophs by acting on
the specific cell surface receptors, thus activating the release of GH.
This released GH in turn acts on muscles, skeleton, and other tissues.

Decreased secretion of growth hormone after real or simulated
weightlessness has been found (7,8,9). An, increased number of growth
hormone granules and hormone content in spaceflight somatotrophs was
observed, suggesting that synthesis was not impaired but that secretion
was (10). Somatotrophic cells harvested

from spaceflight rats did not respond to GRF when implanted into the
lateral brain ventricles of hypophysectomized rats, whereas control
cells did after spaceflight. This observation suggested an impairment in
secretory function (10). Cultured somatotrophs obtained from suspended
rats were also unresponsive when they were challenged with GRF in vitro
(6). Moreover, the decreased secretion of growth hormone as compared to
its formation have been reported after spaceflight (5).

Thus, it was hypothesized that there is an alteration in the number
and/or affinity of GRF receptors, in response to the spaceflight,
causing the impairment of GH secretion.

[Materials and Methods]{.underline}

Methods for our study were adapted form Seifert et al (13).

[Pituitary Homogenates:]{.underline}

Pituitaries from SLS-1 were harvested about 2.5 years ago. Each gland
was individually frozen and stored in 5 ml, individual plastic vials at
-70 degrees centigrade. The glands were markedly desiccated, weighing
about 1/7 that of fresh glands. They averaged 1mg/gland vs. 7-8mg for
fresh glands. On the day of homogenization, the glands were thawed on
ice, pooled by group and presoaked in ice cold Tris Buffer, pH-7.4 (2mM
EGTA, 5mM Mg CL2 and 1% Bovine serum albumin) for two hours. Glands were
then homogenized on ice using a Potter glass homogenizer with teflon
pestle, with final concentration being 10 glands/3.5ml of buffer or 20
mg pituitary tissue/ml of buffer corresponding to 2-3 mg of protein
(measured by the method of Lowry et al). These homogenates were frozen
in plastic tubes at -70 degrees centigrade until assayed.

[Iodinated GRF:]{.underline}

(3-125I-Iodotyrosyl 10) GRF 1-44 amide (human), was obtained from
Amersham, England and stored at -20 degrees centigrade. (Specific
Activity-2000Ci/mmol or 440uCi/ug). One day before assay this iodinated
hormone was diluted with 0.5ml of an acid mix (1:1) of 0.1NHCl and 0.1N
Acetic acid; to this 1.5ml of phosphate buffer were added. Final
dilution was prepared in Tris buffer pH-7.4 up to about 95000cpm/150ul.

[Cold GRF:]{.underline}

Cold GRF (lot no. 138-41-15) was obtained from Dr. Jean Rivier at the
Salk Institute, weighed on Cahn microbalance and diluted in an acid mix
(1:1) of 0.1N HCl and 0.1N acetic acid up to 20uM and frozen at -20
degrees until used. Two weeks later, on the day of assay, the hormone
was thawed on ice, and diluted in Tris buffer to concentrations of:
10-6M, 10-8M, 10-10M, and 10-12M, for use.

[Receptor Assay:]{.underline}

On the day of assay, pituitary homogenates and both iodinated and cold
hormone were thawed on ice and vortexed before use. Assay was performed
in six tubes for each group. One hundred ul of pituitary homogenate
(2-3mg protein) was added to each 12 by 75mm borosilicate glass tube
containing ice cold buffer, followed by 150ul (122 fmol) of iodinated
GRF. Finally, 100ul of each dilution of cold hormone was pipetted into
the tubes. Total reaction volume was 500ul. All tubes were vortexed and
incubated for two hours at room temperature, after which they were
immersed in ice cold water to stop the reaction; 450ul from each tube
were transferred to plastic microfuge tubes, which were then centrifuged
in a tabletop microfuge at 8700 rpm in the cold room. After
centrifugation the supernatant was aspirated and the pellet containing
bottom of tube was cut by using guillotine and placed in plastic tubes
for counting in a Packard Gamma counter.

[Results:]{.underline}

Assays were performed on five groups of glands, including: R+0 control
RAHF, R+0 RAHF flight, Delayed Flight Profile Test (DFPT) R+0 control
RAHF, Delayed Flight Profile Test (DFPT) R+0 RAHF flight, and R+ML
flight RAHF. Total binding was comparable for SLS-1 and fresh glands,
suggesting that total protein was similar, however, there was no
specific binding in any of the flight or control groups. Using the
procedure with freshly harvested glands gave a BMax (capacity) =31.5
fmol/mg protein and Kd (affinity) =0.33nmol. A typical dose-response
curve was obtained from fresh glands showing that the binding of
iodinated GRF to the pituitary receptors was specific and was inhibited
in a dose dependent manner by increasing concentrations of cold GRF.

[Discussion:]{.underline}

Because of the failure to find GRF receptors in five sets of glands, we
did not continue work with the remaining five sets of glands which were
severely desiccated. Results with fresh glands compare well with
Seifert\'s data, but our flight samples did not show specific binding,
suggesting that the receptors had been compromised by desiccation or
other prior treatment of the glands (chilling injury by prolonged freeze
drying). Hence, our question concerning the possible alterations in GRF
receptors in microgravity and the underlying mechanism for the decreased
growth hormone secretion by microgravity remains unanswered.

We continue to believe that this is an important question and that this
study should be repeated using fresher tissues. We also

believe that the persons preserving delicate tissues, such as the
pituitary, should preserve them with consummate care.

[References]{.underline}

1\. Bloch, B., Brazeau, P., Bloom, F., and Ling, N. (1983).
Topographical study of neurons containing hpGRF immunoreactivity in
monkey hypothalamus. Neuroscience Letter. 37:23-28.

2\. Bloch, B., Brazeau, P., Ling, N., Wehrenberg, W.B., Benoit, R.,
Bloom, F., and Guillmin, R. (1983). Immunohistochemical detection of
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3\. Bloch, B., Gaillard, R.C., Brazeau, P., Lin, H.D,. and Ling, N.
(1984). Topographical and ontogenic study of neurons producing growth
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6\. Grindeland, R.E., Mussachia, S.J., Vasques, M.F., and Vale, W.
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bioassayable growth hormone and decreased response to growth hormone
releasing factor. Abstract. ASGSB Annual Mtg., Louisville, KY. p-885.

7\. Grindeland, R.E., Popova, I.A., Vasques, M.F., and Arnaud, B.
(1990). Effects of microgravity on rat body and adrenal weights and
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8\. Grindeland, R.E., (1990). Cosmos 1887: Science Overview. FASEB
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9\. Grindeland, R.E., Ballard, R.W., Collony, J.P., and Vasques, M.
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10\. Hymer, W.C., Grindeland, R.E., Krasnov, I., Victrov, I., Motter,
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11\. Muller, E.E., and Nistico, G. (1989). Brain messenger and the
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12\. Sawchenko, P.E., Swanson, L.W., Rivier, J., and Vale, W. (1985).
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