Fusun Zeytin

Somatocrinin, the growth hormone releasing factor

Publisher Summary This chapter discusses the isolation of tumor-derived and hypothalamic GRFs, structure-activity relationships of synthetic replicates of GRFs, in vitro studies on the mechanism of action of GRF, and antagonism between GRF and somatostatin. Clinical interest in GRF extends over its use as a diagnostic tool and a treatment of hypothalamic dwarfism. GRF helps to promote anabolism in chronic debilitating diseases, as long as the dietary intake is adequate, and to promote the healing of wounds and bone fractures. The availability of GRF with its highly specific effect in stimulating GH secretion should permit investigations of the proposed role of GH in diabetic retinopathy. Structural analogs of GRF acting as competitive antagonists may be of major clinical significance in the treatment of accidents of juvenile diabetes.

Growth hormone-releasing factor: chemistry and physiology.

Conclusions In conclusion, the long-sought growth hormone-releasing factor has been characterized and sequenced in several species (human, porcine, bovine, and murine). The noteworthy achievement with GRF has been the rapidity with which the sum of information has been gathered. In the space of 12 months most of the pioneering studies on the mechanism of action of the peptide in vivo and in vitro were described. Immunocytochemical mapping of GRF neurons was carried out. Structure-function studies were initiated. Clinical trials were started and confirmed the potent GH-releasing activity of GRF in man. The effect of the peptide on specific GH mRNA levels was described and molecular cloning was used to establish the structures of human (pancreas) preproGRF. All the hypothalamic releasing factors which had been postulated in the early fifties as humoral regulators of the secretion of each pituitary hormone have now been characterized.

Somatostatin inhibits growth hormone-releasing factor-stimulated adenylate cyclase activity in GH3 cells

GH3 cells were used to study the effect of rat growth hormone-releasing factor on adenylate cyclase activity and its interaction with somatostatin. Rat GRF stimulates adenylate cyclase activity (ED5 0 = 6 X 10(-8) M) and somatostatin-14 inhibits this GRF-stimulated activity in a non-competitive manner without affecting the basal enzyme levels. Inhibition by somatostatin-14 is observed at concentrations as low as 10(-11) M and the half-maximal effect is obtained with 10(-8) M. Somatostatin-28 is more potent than SS-14 and has an ED5 0 of 3 X 10(-11) M. VIP is more active than rat GRF in stimulating adenylate cyclase activation. We conclude that in GH3 cells rat GRF behaves as a partial VIP agonist by interacting with VIP-preferring receptors and its effects are inhibited by somatostatin.

GRF (somatocrinin) stimulates release of neurotensin, calcitonin and cAMP by a rat C cell line

We reported previously that GRF stimulates release of neurotensin (NT) by a clonal line of rat C cells (44-2 C). We report here that GRF stimulates calcitonin (CT) and cAMP release in these cells. For release experiments, replicate cultures are incubated for 5-180 min in serum-free defined medium. CT, NT and cAMP are measured by RIA. At a maximally effective concentration of GRF (.1-1.0 nM), there is a 2-3 fold stimulation of CT release at 60-90 min with peak release at 180 min. In contrast, GRF causes a rapid 4-6 fold increase of NT release within 5-15 min. In 44-2 C cells there is a 4-40 fold stimulation of cAMP release by GRF. We conclude that in 44-2 C cells GRF stimulates release of NT and cAMP and show for the first time the effect of this peptide on release of CT.

GRF is a highly potent activator of adenylate cyclase in the normal human, bovine and rat pituitary: interaction with somatostatin

The effect of GRF adenylate cyclase activation was studied in normal human, bovine and rat pituitary tissues. Human GRF (hGRF) activates adenylate cyclase in normal human pituitary membrane preparations in a concentration dependent manner (ED5 0 = 10(-11) M). In bovine pituitary cells hGRF stimulates GH secretion into the medium (ED5 0 = 7 X 10(-12) M) and activates adenylate cyclase (ED5 0 = 10(-11) M). In normal rat pituitary cells in monolayer culture, rat GRF (rGRF) stimulates adenylate cyclase (ED5 0 = 3 X 10(-11) M). In normal human pituitary membrane preparations and in normal rat pituitary cells in culture, somatostatin inhibits GRF-stimulated adenylate cyclase in a non-competitive manner, while it does not affect basal (i.e. non-stimulated) adenylate cyclase levels. VIP, a peptide which is structurally homologous to hGRF and rGRF is a weak GRF-agonist and activates adenylate cyclase in human and rat pituitary preparations at concentrations greater than 10 nM.

Rat hypothalamic GRF elicits its biologic action in GH3 cells by interaction with VIP- preferring receptor site(s).

GH3 cells can be used effectively to study the in vitro mechanism of action of GRF. In these cells, there is a time and concentration-dependent release of cAMP into the medium. Rat hypothalamic GRF, (rGRF) is 7 to 10 fold more active than human hypothalamic GRF (hGRF). VIP, a peptide which is structurally homologous to GRF, stimulates cAMP efflux in GH3 cells, with a higher affinity than hGRF or rGRF. We propose that in contradistinction to the normal rat pituitary, the stimulation of cAMP release by GRF in GH3 cells occurs via activation of VIP-preferring receptors and that GRF (rGRF in particular) behaves as a partial VIP agonist.

Somatocrinin stimulates adenylate cyclase-Ns regulatory subunit in a GH3 cell-line: Comparison with VIP

We used a GH3 cell-line to compare the effects of rat GRF (rGRF) and VIP on the adenylate cyclase activity and to determine on what subunit the site of action of these two peptides is. In the GH3 cell-line, VIP was more potent than rGRF to stimulate adenylate cyclase activity. The stimulatory effects of rGRF and forskolin were additive. Cholera toxin decreased the apparent potency of these peptides and pertussis toxin reversed the inhibition by somatostatin of their adenylate cyclase stimulation. We conclude that rGRF acts on the regulatory subunit Ns, different from the regulatory subunit Ni on which somatostatin is suggested to be acting and that, in the GH3 cells, rGRF stimulates adenylate cyclase through VIP-preferring sites.

Effects of forskolin and cholera toxin on cyclic AMP release in a neurotensin-secreting rat C-cell line

The effects of forskolin and cholera toxin on the regulation of cAMP release were studied in a neurotensin-secreting rat C-cell line. The interaction of these agents with norepinephrine, a potent neurotensin secretagogue, was also investigated. Forskolin stimulated cAMP release 10(2)-10(3) fold while it increased neurotensin release 2-3 fold. Cholera toxin caused a 10(2)-10(3) fold increase in cAMP release and had no effect on neurotensin release. We conclude that the 44-2 C-cells provide a new model for studying the regulation of the concomitant (via forskolin) or independent (via cholera toxin) secretion of cyclic AMP and/or neurotensin.