Binding sites for growth hormone releasing factor on rat anterior pituitary cells

Growth hormone releasing factors (GRFs) have been isolated from human pancreatic tumours (hGRF) and rat hypothalamus (rhGRF). The response to GRF at the pituitary level can be modulated by other factors, including glucocorticoids, thyroid hormones, somatostatin and other neuropeptides and somatomedins. Glucocorticoids enhance GRF-induced growth hormone (GH) secretion in primary cultures of rat anterior pituitary cells, and the synthetic glucocorticoid dexamethasone has recently been shown to increase the amounts of GH released in freely moving rats in response to submaximal doses of intravenous GRF. To investigate whether somatotroph sensitivity to GRF is modulated at its receptor level, we have developed a radioreceptor assay using an iodinated analogue of hGRF as radioligand. We report here that the relative binding affinities of rGRF, hGRF and the two analogues are correlated with their in vitro biological potencies. Further, the number of GRF binding sites is drastically decreased in cells deprived of glucocorticoids either in vivo or in vitro.     

Bidirectional control of cytosolic free calcium by thyrotropin-releasing hormone in pituitary cells

It is now established that a key step in the action of calcium-mobilizing agonists is stimulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) to 1,2-diacylglycerol and inositol 1,4,5-trisphosphate (InsP3). The latter substance acts as a second messenger, controlling the release of calcium from intracellular stores (see ref. 3 for review). The bifurcating nature of the signalling system is exemplified by the fact that the other product of PtdIns(4,5)P2 hydrolysis, 1,2-diacylglycerol, can alter cellular function by activating protein kinase C, the cellular target for several tumour-promoting agents such as the phorbol esters. In various tissues, including GH3 pituitary tumour cells, a synergistic interaction between calcium ions and protein kinase C underlies agonist-induced changes in cell activity. The data presented here suggest that when GH3 cells are stimulated by thyrotropin-releasing hormone (TRH), an agonist inducing PtdIns(4,5)P2 hydrolysis, the two limbs of the inositol lipid signalling system interact to control free cytosolic calcium levels [( Ca2+]i). At low levels of TRH receptor occupancy, [Ca2+]i increases rapidly, then declines relatively slowly. As receptor occupancy increases, the calcium signal becomes more short-lived due to the appearance of a second, inhibitory, component. This latter component, which is enhanced when [Ca2+]i is elevated by high potassium depolarization, is mimicked by active phorbol esters and by bacterial phospholipase C. It seems likely that protein kinase C subserves a negative feedback role in agonist-induced calcium mobilization.     

Adenohypophyseal degradation of thyrotropin releasing hormone regulated by thyroid hormones

Thyrotropin Releasing Hormone (TRH; pyroGlu-His-Pro-NH2) is important in the regulation of adenohypophyseal hormone secretion and also serves neurotropic functions in extra-hypothalamic brain areas, indicating that it is involved in neurotransmission and other forms of cellular communication. This hypothesis is strengthened by the observation that TRH is hydrolysed at the pyroGlu-His bond by a particulate enzyme located in the synaptosomal and adenohypophyseal plasma membrane. Furthermore, this enzyme has been identified as a heterogeneously distributed ectoenzyme which has a high degree of substrate specificity like the TRH-degrading serum enzyme studied previously. In the rat, the activity of the TRH-degrading serum enzyme has been shown to be influenced by the thyroid status of the animals; here I report that the activity of the membrane-bound TRH-degrading enzyme of the anterior pituitary is stringently controlled by thyroid hormones, but that the activity of the brain enzyme is not.     

Multiple intracellular signallings involved in regulation of on channels by GH releasing or inhibitory hormones in pituitary somatotropes

Influx of Ca2-via Ca2+ channels is the major step triggering exocytosis of pituitary somatotropes to release growth hormone (GH). Voltage-gated Ca2+ and K+ channels, the primary determinants of the influx of Ca2+ in somatotropes, are regulated by GH-releasing hornone (GHRH) and somatostatin (SRIF) through G protein-coupled signalling systems. Using whole-cell patch-clamp techniques, the changes of the Ca2+ and K+ currents in primary cultured somatotropes were recorded and signalling systems were studied using pharmacological reagents and intracellular dialysis of non-permeable molecules including antibodies and antisense oligonucleotides. GHRH increased both L-and T-types Ca2+ currents and decreased transient (I4) and delayed rectified (Ik) K+ currents. The increase in Ca2+ currents by GHRH was mediated by cAMP/protein kinase A system but the decrease in K+ currents required normal function of protein kinase C system. The GHRH-induced alteration of Ca2+ and K+ currents augments the influx of Ca2+ , leading to an increase in the [ Ca2+ ]I and the GH secretion. In contrary, a significant reduction in Ca2+ currents and increase in K currents were obtained in response to SRIF. The ion channel response to SRIF was demonstrated as a membrane delimited pathway and can be recorded by classic whole-cell configuration, Intracellular dialysis of anti-αi3 antibodies attenuated the increase in K + currents by SRIF whereas anti-αo antibodies blocked the reduction in the Ca2+ current by SRIF. Dialysis of antisense oligonucleotides specific for αo2 sub-units also attenuated the inhibition of SRIF on the Ca2+current. The Gi3 protein mediated the increase in K + currents and the Go2 protein mediated the reduction in the Ca2 +current by SRIF. The SRIF-induced alteration of Ca2 + and K + currents diminished the influx of Ca2+ , leading to a decrease in the [ Ca2+ ]I and the GH secretion. It is therefore concluded that multiple signalling systems are employed in the ion channel response to GHRH or SRIF in somatotropes, which leads to an increase or decrease in the GH secretion.