IMMORTALIZED HYPOTHALAMIC LUTEINIZING-HORMONE-RELEASING HORMONE (LHRH) NEURONS - A NEW TOOL FOR DISSECTING THE MOLECULAR AND CELLULAR BASISOF LHRH PHYSIOLOGY
Wc. Wetsel, IMMORTALIZED HYPOTHALAMIC LUTEINIZING-HORMONE-RELEASING HORMONE (LHRH) NEURONS - A NEW TOOL FOR DISSECTING THE MOLECULAR AND CELLULAR BASISOF LHRH PHYSIOLOGY, Cellular and molecular neurobiology, 15(1), 1995, pp. 43-78
1. Two LHRH neuronal cell lines were developed by targeted tumorigenes
is of LHRH neurons in vivo. These cell lines (GN and GT-1 cells) repre
sent a homogeneous population of neurons. GT-1 cells have been further
subcloned to produce the GT1-1, GT1-3, and GT1-7 cell lines. While co
nsiderable information is accumulating about GT-1 cells, very little i
s currently known about the characteristics and responses of GN cells.
2. By both morphological and biochemical criteria, GT-1 cells are cle
arly neurons. All GT-1 cells immunostain for LHRH and the levels of pr
ohormone, peptide intermediates, and LHRH in the cells and medium are
relatively high. 3. GT-1 cells biosynthesize, process, and secrete LHR
H. Processing of pro-LHRH appears to be very similar to that reported
for LHRH neurons in vivo. At least four enzymes may be involved in pro
cessing the prohormone to LHRH. 4. LHRH neurons are unique among the n
eurons of the central nervous system because they arise from the olfac
tory placode and grow back into the preoptic-anterior hypothalamic reg
ion of the brain. Once these neurons reach this location, they send th
eir axons to the median eminence. With respect to the immortalized neu
rons, GN cells were arrested during their transit to the brain. In con
trast, GT-1 cells were able to migrate to the preoptic-anterior hypoth
alamic region but were unable correctly to target their axons to the m
edian eminence. These problems in migration and targeting appear to be
due to expression of the simian virus T-antigen. 5. While GT-1 cells
are a homogeneous population of neurons, they are amenable to cocultur
e with other types of cells. Coculture experiments currently under way
should help not only to reveal some of the molecular and cellular cue
s that are important for neuronal migration and axonal targeting, but
they should also highlight the nature of the cellular interactions whi
ch normally occur in situ. 6. GT-1 cells spontaneously secrete LHRH in
a pulsatile manner. The interpulse interval for LHRH from these cells
is almost identical to that reported for release of LH and LHRH in vi
vo. GT-1 cells are interconnected by both gap junctions and synapses.
The coordination and synchronization of secretion from these cells cou
ld occur through these interconnections. by feedback from LHRH itself,
and/or by several different compounds that are secreted by these cell
s. One such compound is nitric oxide. 7. GT-1 cells have Na+, K+, Ca2, and Cl- channels. Polymerase chain reaction experiments coupled with
Southern blotting and electrophysiological recordings reveal that GT-
1 cells contain at least five types of Ca2+ channels. R-type Ca2+ chan
nels appear to be the most common type of channel and this channel is
activated by phorbol esters in the GT-1 cells. 8. LHRH is secreted fro
m GT-1 cells in response to norepinephrine, dopamine, histamine, GABA
(GABA-A agonists), glutamate, nitric oxide, neuropeptide Y, endothelin
, prostaglandin E(2), and activin A. Phorbol esters are very potent st
imulators of LHRH secretion. Inhibition of LHRH release occurs in resp
onse to LHRH, GABA (GABA-B agonists), prolactin, and glucocorticoids.
9. Compared to secretion studies, far fewer agents have been tested fo
r their effects on gene expression. All of the agents which have been
tested so far have been found either to repress LHRH gene expression o
r to have no effect. The agents which have been reported to repress LH
RH steady-state mRNA levels include LHRH, prolactin, glucocorticoids,
nitric oxide, and phorbol esters. While forskolin stimulates LHRH secr
etion, it does not appear to have any effect on LHRH mRNA levels.