Hormonal regulation of cytosolic free calcium and its functional consequences: the GH-cell model.

Use of clonal strains of prolactin (PRL)- and growth hormone-producing rat pituitary cells has proven informative in elucidating a number of the early biochemical, ionic, and secretory events regulated by the hypothalamic tripeptide, thyrotropin-releasing hormone (TRH). TRH causes biphasic changes in the concentration of cytosolic free calcium [( Ca2+]i) in GH4C1 cells and biphasic changes in hormone secretion. Early changes occur on a msecond to second scale and late changes, on a time scale of minutes. Although increases in [Ca2+]i are essential for enhanced secretion, at least in the case of the rapid initial phase, the TRH-induced increase in [Ca2+]i is necessary, but not sufficient to enhance secretion. A co-mediator with calcium appears to be diacylglycerol. The majority of the calcium involved in the early phase of rise in [Ca2+]i induced by TRH is derived from intracellular sources, while essentially all of the calcium rise observed in the late phase is derived from extracellular calcium entering the cell through both voltage-dependent and voltage-independent conductances. Because TRH causes an elevation of inositol(1,4,5) trisphosphate [Ins(1,4,5)P3] within seconds, but not mseconds, further studies are required before it can be concluded unequivocally that Ins(1,4,5)P3 is the sole mediator of the rapid phase of rise in [Ca2+]i induced by TRH in GH-cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Introduction
This paper summarizes the results of experiments performed in my own laboratory on the regulation of cytosolic free calcium [Ca2+]i and prolactin (PRL) secretion by thyrotropin-releasing hormone (TRH). The model we have investigated extensively uses the so-called GH-cell system, particularly GH4C1 and GH3 cells (1)(2)(3). These cells have been used extensively in many laboratories worldwide to study the regulation of the expression of the growth hormone and PRL genes, the mechanisms of action of vasoactive intestinal peptide, somatostatin, epidermal growth factor, platelet-derived growth factor, bombesin, thyroid hormones, glucocorticoids, estradiol, and 1,25(OH) 2cholecalciferol (1,25(OH)2D3) as well as the phorbol ester tumor promoters. Furthermore, a number of studies have used electrophysiological methods to characterize ion channels and cation conductances *Laboratory of Toxicology, Harvard School of Public Health and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115. across the plasma membrane of these cells. In this synopsis I shall not cite any of the many and important contributions from other investigators. I have chosen to discuss several specific aspects of the roles of [Ca2+]i and inositol phospholipids in hormone-receptor signaling pathways in GH-cells. A number of generalities can be derived from this cell system, but it is not representative of all proteinsecreting cells that use inositol lipids and their hydrolysis products for intracellular signaling. This conclusion does not, however, limit the usefulness of GH-cells but indicates that other models need to be studied in comparable detail.
Early Actions of TRH on Cytosolic Free Calcium The actions of TRH on GH4C1 cells mediated via its specific plasma membrane receptor (4-6) that occur over a time span of several hundred mseconds to several minutes include the following: a) increases in [Ca2+]i that are biphasic, b) enhanced inositol lipid hydrolysis, c) release of cell calcium into the extracellular environment, d) influx of extracellular calcium, e) enhanced number of calcium-dependent action potentials, ) activation of protein kinase C and other protein kinases, g) altered pattern of protein phosphorylation, h) increased intracellular pH (pHi), and i) release of stored, previously synthesized PRL that is biphasic.
Three major experimental approaches have been used to examine the early actions of TRH. These are electrophysiological studies, 'Ca2" flux analyses, and biochemical investigations of phospholipid turnover and protein phosphorylation.  (8,9). An initial burst of PRL release occurs within the first 1 to 2 min, which is reflected in a 10to 20-fold increase in the interval rate of secretion. Thereafter, the rate of TRH-enhanced secretion falls to a new sustained rate that is 2to 3-fold above the control rate. We have performed analogous experiments with calcium channel antagonists that block voltagedependent calcium channels in GH4C1 cells (8). Nifedipine  Based on knowledge from other laboratories that TRH activates phospholipase C via its receptor and a G-protein, we consider it likely that diacylglycerol (DAG) acts as a co-mediator with calcium in the burst phase of PRL secretion. This conclusion is supported by the results of two kinds of experiments. First, we reconstituted the precise TRH-induced pattern of change in both [Ca2+]i and PRL secretion using ionomycin to cause the spike in [Ca2+]i plus the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to mimic the action of endogenous DAG (9). When secretion was examined, the TRH-induced pattern was also reproduced, essentially identically, by the combination of ionomycin and TPA (9). The second line of evidence implicating endogenous DAG in the acute secretory action of TRH came from experiments examining the rapid activation of protein kinase C by TRH (11). We measured activation of protein kinase C by determining its subcellular localization at early times after treating GH4C1 cells with TRH (11). There was a rapid (< 10 sec) loss of protein kinase C activity or phorbol ester binding from the cytosol and also an equally rapid increase in protein kinase C in the particulate membrane fraction of the cell. Thus, it was possible to mimic TRH actions on [Ca2+]i and secretion with ionomycin plus TPA and demonstrate that TRH activates rapidly protein kinase C, an important intracellular target for the action of endogenous DAG.
Results from several other investigators have shown that TRH activates phospholipase C and causes generation of Ins(1,4,5)P3 in GH-cells within 2 to 5 sec. Ins(1,4,5)P3 also causes calcium release from sequestered stores when added to permeabilized GHcells. However, as indicated above, TRH causes an elevation of [Ca2+]i in GH-cells within 300 msec (8). If Ins (1,4,5)P3 that is generated in response to TRH is the intracellular mediator of TRH action on [Ca2+]j, it would be expected that enhanced Ins(1,4,5)P3 formation would occur as rapidly, if not more rapidly, than the increase in [Ca2+]i induced by TRH. When we examined the kinetics of Ins(1,4,5)P3 in detail in GH4C1 cells, we found a lag of 1000 to 1200 msec before the increase in Ins(1,4,5)P3 could be detected, while the rise in [Ca2+]i occurred within about 300 msec (12). This result was not due to our inability to measure rapid changes in inositol polyphosphates, because we could measure changes in InsP4 and InsP5 between 400 and 1000 msec (12,13). These findings suggest that the current dogma that Ins (1,4,5)P3 is the sole mediator of intracellular calcium redistribution in GH-cells may need to be reconsidered.

Additional Aspects of Signal Transduction in GH-CelIs
In the course of further studies with ionomycin, C.W. Fearon noted that low concentrations of the ionophore itself did not cause redistribution of protein kinase C, but that pretreatment with ionomycin antagonized the action of TRH (14). The antagonism required pretreatment for greater than 10 sec and was reversed by high K+ in the presence of extracellular calcium. These results lead us to conclude that optimal activation of protein kinase C by TRH requires a simultaneous rise in both DAG and [Ca2]i (14). GH4C1 cells were loaded with Quin 2 and the fluorescent proton reporter bis(carboxyethyl)carboxy-fluorescein (BCECF) and TRH-induced changes in pHi monitored (15). In acid loaded cells, TRH causes a rapid increase in pHi that is independent of changes in [Ca2+]i and is blocked by amiloride. Thus, TRH stimulates the activity in the Na+/H+ antiporter in GH-cells. The mechanism appears to involve activation of protein kinase C, but this may not be the sole mode of control of pHi by TRH (18). The mechanism does not involve a change in TRH receptors, in inositol phosphate generation and in protein kinase C activity, or an increase in the intracellular reservoir of calcium on which TRH acts. Because acute removal of extracellular calcium blocks the 1,25(OH)2D3 effect, the action of vitamin D appears to be mediated by enhanced influx of extracellular calcium induced by TRH (18). The TRHregulated influx channel or pathway regulated by 1,25(OH)2D3 has not yet been identified. Finally, we have solubilized the TRH receptor from GH-cells using 1% digitonin (19). Solubilization of the unoccupied receptor gives a soluble receptor that preserves the binding characteristics of the membrane or whole cell receptor including competition for TRH binding by chlordiazepoxide. Solubilization of the TRH-occupied receptor gives a soluble and functional TRH-receptor-G protein complex. Conclusion GH-cells have proven useful in identifying and characterizing a variety of important receptor-mediated transduction and regulatory pathways in protein hormone secreting cells. Although much has been learned, much more remains to be discovered using this novel cell culture system.
The experiments described in this resume were performed under support, in part, by a research grant from NIH (DK 11011). The author thanks Jean Foley for her expert help in preparing this manuscript.