Construction of the Model Construction of the Model
To construct the model two
important methodologies have been used, immunocytochemistry and superfusions.
Immunocytochemistry has been use to identify potential regulatory factors in the
neurointermediate lobe. The entire neurointermediate lobe has been considered in the
search for potential secretagogues. The neural lobe is rich in nerve terminals
containing a variety of neuropeptides. The vascularization of the pars intermedia comes
from the pars nervosa and thus anything released from terminals in the pars nervosa can
easily reach the pars intermedia, either through the blood or diffusion through the
extracellular space.
The antibodies used in these studies have been against either the potential regulatory factors themselves (e.g. serotonin, CRH, TRH) or against enzymes essential for the biosynthesis of the transmitter substance (e.g. antibodies against tyrosine hydroxylase (TH), an essential enzyme for biosynthesis the catecholamines, or antibodies against glutamic acid decarboxylase (GAD), a marker enzyme for GABAergic neurons).
Having identified potential
regulatory factors, or secretagogue, with the above approach the next question to be
examined was if the factor is indeed involved in regulating the secretory activity of the
melanotrope cell. To answer this question superfusion (also called perifusion) methods
have been used. Here neurointermediate lobes of Xenopus are placed in small
superfusion chambers (volume 10 ml)
and incubation medium is pumped through the chamber and collected in a fraction collector.
Each fraction is then submitted to a radioimmunoassay for a-MSH to determine how much peptide was secreted
to the incubation medium. The potential regulatory factors are introduced into the medium
being pumped over the lobes and thus, with the radioimmunoassay, it is possible to
determine if the factor effects MSH secretion, either in a stimulatory or inhibitory
fashion.
The above studies are with
whole tissue and thus it can not be ruled out any effects seen on secretion could be
indirect (e.g. stimulating or inhibiting release of transmitters from nerve
terminals in the tissue). The radioimmunoassay proved to be too insensitive to measure a-MSH secretion from isolated melanotrope cells.
For this reason another method was developed where secretion could be studied from
isolated and cultured melanotrope cells. This method involved incubating freshly
isolated melanotrope cells in incubation medium containing radioactive amino acids (e.g.
3H-lysine) for 2 days. During the 2 days the cells attach to the glass cover slips on
which they are cultured and they incorporate the radioactive amino acids into
proopiomelanocortin (POMC), the precursor protein for a-MSH. This precursor is subsequently processed to produce radiolabeled a-MSH and other peptides derived from POMC.
The cell preparations can then be submitted to superfusion and the medium collected and
the radioactivity in each fraction determined in a b-counter.
The above superfusion methods (with neurointermediate lobe tissue and with cultured melanotrope cells) have been used to identify the receptor mechanisms utilized by the various regulatory factors. The response of the tissue or cells to specific receptor agonists and antagonists is determined. In this way we have shown that e.g. dopamine inhibits through the D2 receptors and GABA through both a GABAa and a GABAb receptor.
