Differential effects of thyrotropin-releasing hormone, vasoactive intestinal peptide, phorbol ester, and depolarization in GH4C1 rat pituitary cells

The sequence of PRL and GH release from GH4C1 cells was studied in perifusion and static culture systems. The secretory pattern elicited by TRH differed from those caused by depolarizing concentrations of KCl (Ca2+-initiated secretion), vasoactive intestinal peptide (VIP), 8-bromo-cAMP, and forskolin (cAMP-mediated secretion), and 12-O-tetradecanoylphorbol-13-acetate (TPA) (protein kinase C activation). TRH, K+, VIP, and TPA all caused secretion within 1 min in the perifusion system but the peak response to TRH and depolarization occurred earlier than the peak responses to TPA and VIP. PRL and GH release in response to a pulsatile application of TRH (0.4-min pulse every 5 min for 25 min) did not decline with a low dose, indicating that acute desensitization does not occur, but did decrease with a high concentration. When cells in the perifusion system were subjected to continuous stimulation, TRH caused a biphasic response with a 2- to 3-min period of high secretion followed by a second phase in which GH and PRL secretion were 60-70% the rates in the first phase. KCl caused predominantly first-phase secretion, and TPA caused a biphasic secretory pattern with a delay in its peak of action. VIP caused a modest but prolonged response whether administered in a pulsatile or sustained manner. When GH-cells were exposed to 100 nM TRH for 2 days, [3H] [N3-methyl-His2]TRH binding was decreased (down-regulation), intracellular PRL was increased (170% of control), and intracellular GH was decreased (65% of control). In these down-regulated cells, baseline PRL and GH secretion were changed in proportion to the relative intracellular hormone content. The responsiveness to TRH, KCl, and TPA during the initial 10-min period (first phase) was reduced; however, the responsiveness to these substances in the subsequent 50-min period (second phase) was unchanged. The ED50 for TRH stimulation of hormone release was increased 2- to 4-fold in down-regulated cells, but the dose-response curves for other secretagogues were not shifted. These data suggest that the initial burst of hormone release caused by TRH is mediated by Ca2+, and that prolonged exposure to TRH causes homologous desensitization.     

Degradation of thyrotropin-releasing hormone by the GH3 strain of pituitary cells in culture.

Thyrotropin-releasing hormone (TRH), pGlu-His-ProNH2, binds within 1 h to specific receptors on the GH3 strain of pituitary cells. When GH3 cells were incubated for 2 days with 3 nM [2,3-3H-Pro]TRH, an increasing fraction of the total cellular radioactivity (7% after 1 h, 81% after 43 h) was associated covalently with proteins as determined by dialysis, acid precipitation, and gel filtration; this fraction corresponded to label which could not be displaced from intact GH3 cells by the addition of excess unlabeled TRH. R5 and GH12C1 cells, strains which lack TRH receptors, accumulated 16 or 23%, respectively, as much label from [2,3-3H-Pro]TRH as did GH3 cells in 24 h. After 24 h of incubation with [2,3-3H-Pro]TRH and [14C-His]TRH, the ratio of 14C/3H in GH3 cells was the same as in the culture medium, indicating that the intact tripeptide was taken up by the cells. After 24-48 h of incubation with [2,3-3H-Pro]TRH, GH3 proteins appeared to be labeled randomly as surmised by fractionation on Sephadex G-100, DEAE cellulose, Sepharose 4B and sucrose density gradients. In cultures treated with cycloheximide (10 mug/ml) or proline (6.3 mM) the initial binding of [2,3-3H-Pro]TRH to receptors, measured after 1 h, was 97% or 102% of control. However, the incorporation of label from [2,3-3H-Pro]TRH into an acid-precipitable product after 22 h was inhibited by 81 and 74% by cycloheximide (1 mug/ml) and proline (2.5 mM). Formation of [2,3-3H] proline from [2,3-?3H-Pro] TRH was demonstrated by thin layer chromatography; the percentage of non-protein radioactivity with an Rf of proline increased from 20 to 80% in GH3 cells incubated 1 or 24 h with [2,3-3H-Pro]TRH. We conclude that after binding to receptors on GH3 cells, TRH is slowly metabolized to its constituent amino acids, and the products [2,3-3H]proline or [14C]histidine are incorporated into newly synthesized proteins.     

Relationships between thyroid hormone and glucocorticoid effects in GH3 pituitary cells

The interrelationships between thyroid hormone and cortisol actions were investigated in GH3 pituitary tumor cells. When GH3 cells were grown in thyroid hormone-deficient medium, cortisol did not affect the concentration of TRH receptors. Both thyroid hormones and TRH normally decrease the number of TRH receptors, and cortisol inhibited down-regulation by both hormones. TRH caused a greater increase in PRL synthesis when TRH receptors were high in the presence of cortisol and T3 than when TRH receptors were low (T3 alone). In the presence of cortisol, higher concentrations of T3 were required to decrease TRH receptors, while lower concentrations were necessary to stimulate GH synthesis. Cortisol and T3 alone stimulated GH synthesis 6- and 10-fold, respectively, while together they caused an 830-fold increase. In contrast, T3 did not alter the inhibition of PRL synthesis by the glucocorticoid. Cortisol did not significantly affect the amount of [125I]T3 bound to nuclei from cells incubated in thyroid hormone-deficient or T3-supplemented medium (approximately 100 and approximately 25 fmol/mg cell protein). The data suggest that cortisol modifies thyroid hormone action at a step subsequent to T3 receptor binding.     

Benzodiazepines modulate voltage-sensitive calcium channels in GH3 pituitary cells at sites distinct from thyrotropin-releasing hormone receptors

Benzodiazepines (BZs) have been shown to modulate voltage-sensitive Ca2+ channels in a number of neuronal and nonneuronal cell types and to competitively antagonize TRH binding to receptors on cells of the nervous system and anterior pituitary gland. Because interaction of TRH with its receptor is known to cause enhanced influx of Ca2+ through voltage-sensitive channels in rat pituitary GH3 cells, it was determined whether BZs and TRH were interacting with the same binding site on these cells. The potencies of three BZs, Ro5-4864, diazepam (DZP), and chlordiazepoxide (CDE), were compared as modulators of Ca2+ channels and as inhibitors of TRH binding in GH3 cells. Modulation of Ca2+ channel activity was measured as the inhibition by BZs of K+ depolarization-induced Ca2+ influx using intracellularly trapped quin 2 or 45Ca2+ uptake. The three BZs caused dose-dependent inhibition of Ca2+ influx with an order of potency of Ro 5-4864 greater than DZP greater than CDE. In contrast, the order of potency of the three BZs to inhibit [3H]TRH binding was CDE greater than DZP much greater than Ro 5-4864. The concentrations of BZs needed to inhibit Ca2+ influx and TRH binding were in the micromolar range. These data show that BZs can modulate Ca2+ channel activity in endocrine cells and that these sites are distinct from those that modulate TRH binding on pituitary cells.     

Reevaluation of the electrophysiological actions of thyrotropin-releasing hormone in a rat pituitary cell line (GH3)

The electrophysiological actions of TRH were examined in the clonal pituitary cell line GH3 with the use of the perforated patch variation of the standard whole cell patch-clamp technique. The action of TRH on spontaneously spiking cells was to cause a brief hyperpolarization (first phase action), followed by a period during which action potential behavior was significantly modified (second phase action). The modifications during second phase action included a reduction in the slope of the up-stroke, a reduced peak potential, an increase in duration, and a depolarizing shift of the after-hyperpolarization. The modification of voltage- and calcium-dependent conductances that underlie these changes were investigated in voltage clamp experiments. During first phase action TRH was found to increase calcium-dependent potassium current. During second phase action TRH was found to significantly reduce the L-type calcium current (35%), with no alteration in the T-type calcium current. The second phase action of TRH on calcium-dependent potassium conductance was complex. First, a decrease was observed. This was followed by an increase that did not become fully manifest until after TRH was washed from the cell. TRH caused no change in voltage-dependent potassium current. These results indicate that the second phase action of TRH on action potential behavior in GH3 cells is mediated by a reduction in L-type calcium current and alterations in the behavior of calcium-dependent potassium currents, but not through changes in voltage-dependent potassium currents.     

Arginine increases growth hormone gene expression in rat pituitary and GH3 cells

The effect of arginine (Arg) and Ornitargin (OT) [a compound containing the aminoacids Arg, citrulline (Cit) and ornithine (Orn)] administration upon growth hormone (GH) gene expression was studied both in vivo and in vitro (hemipituitaries and GH3 cells) by Northern blot analysis. For in vivo studies, adult male Wistar rats were anesthetized, subjected to i.v. infusion of 200 microl of 150 mM NaCl (control group), Arg (15 or 150 mg) or OT (15 mg of Arg, 1 mg of Cit and 4 mg of Orn) at a rate of 20 microl/min, and killed 50 min thereafter. For the in vitro studies, hemipituitaries or GH3 cells were incubated in 1 ml of appropriate medium containing Arg (15 or 150 mg) or OT (15 mg of Arg, 1 mg of Cit and 4 mg of Orn) for 60 min. The pituitaries of the in vivo and in vitro studies and GH3 cells were subsequently processed for RNA extraction. Total RNA was subjected to electrophoresis in agarose (1%)/formaldehyde gel, transferred to a nylon membrane and subjected to hybridization with a rat GH (32)P-cDNA, and (32)P-18S rRNA probe to correct for the variability in RNA loading. After autoradiography of the membrane, the abundance of GH mRNA and 18S rRNA bands was quantified by densitometry. The in vivo study demonstrated that Arg and OT infusion induced a 2.3-fold increase in GH mRNA expression, which could result from the Arg-mediated inhibition of somatostatin release. In addition, in vitro Arg, but not OT, induced GH gene expression in hemipituitaries and GH3 cells, indicating that the aminoacid can act per se at the pituitary somatotrope level. In conclusion, our data show for the first time that arginine stimulates GH gene expression in parallel to its recognized GH-releasing activity.     

Acute stimulated hormone release from cultured GH3 pituitary cells.

Treatment of cultured rat pituitary GH3 cells with 50 mM KCl in growth medium released 33% of cell PRL and 18% of cell GH with a half-time of 5 min. Hormone in the culture medium was increased 2- to 4-fold over unstimulated levels. The response required calcium; barium and strontium, but not magnesium, could substitute for calcium. Low temperature completely inhibited hormone release, which was also reduced significantly by inhibitors of energy metabolism and by nitrogen. This acute response was similar in ionic requirements, hormones released, and time course to the acute effect of TRH. Like potassium stimulation, TRH resulted in acute release of both PRL and GH. This contrasts with the finding that chronic TRH treatment reduced GH synthesis in GH3 cells. After a 10-min preincubation with potassium, subsequent short incubations with potassium released little hormone unless the cells were allowed to recover by incubation in normal medium for at least 2 h. This acutely releasable hormone pool seems to be located in a membrane-bound subcellular fraction, since GH3 cells did not discharge the cytoplasmic marker enzyme, lactate dehydrogenase, during potassium-stimulated hormone release.     

Ceramide enhances growth hormone (GH)-releasing hormone-stimulated cyclic adenosine 3',5'-monophosphate accumulation but inhibits GH release in rat anterior pituitary cells

In this study, the effect of ceramide on GH-releasing hormone (GHRH)-stimulated cAMP accumulation and GH release in rat anterior pituitary cells was investigated. C2-, C6-, and C8-ceramide were found to enhance GHRH-stimulated cAMP accumulation. In contrast, their effects on GHRH-stimulated GH release were inhibitory. Treatment with a glucosylceramide synthase inhibitor produced a similar enhancing effect on cAMP accumulation and an inhibitory effect on GH release. To identify the pathway through which ceramide mediated its effect, it was found that ceramide inhibited GH release stimulated by KCl, BayK 8644, and a GH-releasing peptide, but not that stimulated by ionomycin or an activator of protein kinase C. Direct measurement of intracellular Ca2+ revealed that C2-ceramide inhibited GHRH- and KCl-mediated increases in intracellular Ca2+, suggesting that ceramide probably inhibits GH release through inhibition of the L-type Ca2+ channels. As for its mechanism on cAMP accumulation, the enhancing effect of ceramide on GHRH-stimulated cAMP accumulation was abolished in the presence of a phosphodiesterase inhibitor, isobutylmethylxanthine, suggesting that ceramide enhances the cAMP response through inhibition of its metabolism. Taken together, our results suggest that ceramide plays an important role in the regulation of GHRH-stimulated responses in somatotrophs. By reducing GH secretion while enhancing cAMP accumulation, ceramide may promote the synthesis and storage of GH in rat anterior pituitary cells.     

Prolonged stimulation with thyrotropin-releasing hormone and pituitary adenylate cyclase-activating polypeptide desensitize their receptor functions in prolactin-producing GH3 cells

We used somatolactotroph GH3 cells to examine changes in response to stimulation with thyrotropin-releasing hormone (TRH) and pituitary adenylate cyclase-activating polypeptide (PACAP) after sustained treatment with these peptides. TRH and PACAP increased prolactin promoter activity in mock- and PACAP type 1 receptor (PAC1R)-transfected cells. When the cells were pretreated with TRH for 48 h, the response of the prolactin promoter to both TRH and PACAP was diminished. Similarly, in PAC1R-transfected GH3 cells pretreated with PACAP, the effects of TRH and PACAP on the prolactin promoter were eliminated. The stimulation of prolactin mRNA expression by TRH and PACAP was eliminated by prolonged pretreatment with these peptides in PAC1R-transfected cells. Both the serum response element (SRE) promoters and cAMP response element (CRE) promoters were activated by TRH and PACAP in either mock- or PAC1R-transfected cells. Pretreatment for 48 h with TRH also eliminated the effects of TRH and PACAP on the SRE and CRE promoters, and pretreatment of PAC1R-transfected cells with PACAP for 48 h reduced the responses of the SRE and CRE promoters to TRH and PACAP. These observations demonstrated that sustained stimulation with TRH and PACAP desensitizes their own and each other’s receptors.     

Thyrotropin-releasing hormone stimulates GTP hydrolysis by membranes from GH4C1 rat pituitary tumor cells.

Thyrotropin-releasing hormone (TRH) stimulates prolactin production by GH4C1 rat pituitary tumor cells, which possess high-affinity membrane receptors for the peptide. TRH caused up to a 50% increase in the activity of a low-Km GTPase in membranes from GH4C1 cells. The TRH stimulatory effect was maximal at GTP concentrations of 1 microM or lower. TRH caused an increase in GTPase activity of between 0.2 and 20 pmol of GTP hydrolyzed per mg of protein per min, depending on GTP concentration, while TRH binding was 0.3 pmol/mg of protein. TRH did not stimulate GTPase activity in membranes from GH12C1, or GH-Y cells, two pituitary lines lacking TRH receptors. Stimulation of GTPase depended on occupancy of the TRH receptor; half-maximal increases in GTPase activity required 46 nM TRH and 25 nM [N3-methyl-His]TRH, but the TRH free acid was inactive. The apparent Kds of these peptides for receptors were similar when measured under the same conditions. The fact that TRH binding to receptors is regulated by guanyl nucleotides, together with the demonstration of TRH stimulation of low-Km GTPase activity, suggests that the TRH receptor is associated with a guanyl nucleotide regulatory protein in the lactotroph membrane.     

Evidence for tight coupling of receptor occupancy by thyrotropin-releasing hormone to phospholipase C-mediated phosphoinositide hydrolysis in rat pituitary cells: use of chlordiazepoxide as a competitive antagonist

Chlordiazepoxide (CDE) has been shown to antagonize the effects of TRH to stimulate the hydrolysis of phosphoinositides and elevate cytoplasmic free calcium in rat pituitary tumor (GH3) cells. Herein, we show that CDE inhibits TRH stimulation of PRL secretion and that the effect of CDE to antagonize TRH action is caused by its ability to compete with TRH for binding to receptors on GH3 cells. We also use CDE to explore whether continued receptor occupancy is required for prolonged stimulation of cellular responses. CDE had no effect on basal PRL secretion, but caused a dose-dependent inhibition of TRH-induced PRL secretion. CDE decreased the affinity of TRH binding to intact GH3 cells without affecting the maximum binding capacity. As shown previously, CDE had no effect on phosphoinositide metabolism, which was monitored because it appears to be a mechanism for signal transduction by TRH, and when added simultaneously with TRH, caused a dose-dependent inhibition of TRH-induced phosphoinositide metabolism. When CDE was added to cells 2.5 or 5 min after TRH, CDE rapidly terminated the stimulation by TRH of phosphoinositide hydrolysis, shown as inhibition of the continued formation of inositol phosphates and inositol, and of the decrease in phosphoinositides. Lastly, when cells were stimulated with 50 nM TRH, then exposed to 100 microM CDE, and finally to 1000 nM TRH, inositol phosphate formation was stimulated, then inhibited, and then restimulated. These data demonstrate that CDE acts as a competitive antagonist of TRH action on GH3 cells by competing with TRH for binding to its receptor and that continued stimulation by TRH of phospholipase C-mediated hydrolysis of phosphoinositides is tightly coupled to receptor occupancy.     

Regulation of thyroid hormone receptors and responses by thyrotropin-releasing hormone in GH4C1 cells

The present study was undertaken to test the effects of TRH on thyroid hormone receptors and responses in GH4C1 rat pituitary tumor cells. TRH caused a loss of up to 32% of specific nuclear thyroid hormone binding sites with an ED50 of approximately 1 nM, and this loss was additive to the receptor down-regulation caused by T3 itself. Scatchard analysis of nuclear T3 binding revealed that 10 nM TRH decreased the concentration of T3 receptors from Bmax (femtomoles per mg protein) of 110 to 50 while receptor affinity in serum-free medium changed from dissociation constant (Kd) 110 to 50 pM with TRH. TRH lowered the GH response to 0.5 nM T3 from 215% to 127% of control. The concentrations of TRH required to decrease T3 receptors and T3 responses were similar and indicated that these TRH effects are mediated by the TRH receptor. In the absence of added thyroid hormone TRH had little effect on the rate of GH synthesis. TRH did not affect the binding of 0.5 nM [125I]T3 to receptors during the first 8 h but reduced T3 receptor occupancy up to 25-50% in different experiments after 24 h. TRH blocked the induction of GH by T3 only after 48 h or longer. When cells were incubated for 2 weeks with or without 2 nM T3 and 10 nM TRH, the stimulation of cell growth by T3 was decreased by TRH (2- vs. 5-fold increase in cell number) as was stimulation of GH by T3 (5- vs. 13-fold). As expected, T3 blunted the PRL response to TRH from 19- to 3-fold. The effects of TRH on the density of thyroid hormone receptors could be mimicked by the calcium channel agonist BAY K8644 plus a protein kinase C-activating phorbol ester which together caused a 53% reduction in thyroid hormone binding. The dose-response and temporal relationships suggest a causal relationship between the TRH-mediated decrease in thyroid hormone receptors and the decrease in thyroid hormone responses in GH4C1 cells. It has previously been shown that thyroid hormones decrease the concentration of TRH receptors and TRH responsivity in pituitary cells. The results shown here for GH4C1 cells suggest that TRH regulation of T3 responses may also be important in feedback control at the pituitary level.     

Evidence for regulation of a thyrotropin-releasing hormone degradation pathway in GH3 cells

GH3 cells, cloned from a rat anterior pituitary tumor, synthesize and secrete PRL in response to TRH. One of the pathways of TRH degradation is removal of the N-terminal pyroglutamyl residue catalyzed by pyroglutamyl peptide hydrolase (PPH; EC 3.4.11.8). We recently described the synthesis and properties of 5-oxoprolinal, a specific and potent (Ki = 26 nM) inhibitor of PPH. The effect of long term exposure of GH3 cells to 5-oxoprolinal on PPH activity was studied by incubating cells with inhibitor for 3 days, harvesting, washing to remove inhibitor, and assaying for PPH. Unexpectedly, we found a marked (300%) increase in PPH activity. This effect was dependent on the concentration of 5-oxoprolinal (EC50 = 10(-7) M) and was time dependent, with a rapid increase in enzyme activity occurring during the first 24 h. Cycloheximide did not block the increase. The results suggest that the activity of PPH in GH3 cells is subject to complex regulatory mechanisms.     

Gi2 and protein kinase C are required for thyrotropin-releasing hormone-induced stimulation of voltage-dependent Ca2+ channels in rat pituitary GH3 cells.

In rat pituitary GH3 cells, thyrotropin-releasing hormone (TRH) and other secretion-stimulating hormones trigger an increase in the cytosolic Ca2+ concentration by two mechanisms. Ca2+ is released from intracellular stores in response to inositol 1,4,5-trisphosphate and can enter the cell through voltage-dependent L-type Ca2+ channels. Stimulation of these channels is sensitive to pertussis toxin, indicating that a pertussis toxin-sensitive heterotrimeric guanine nucleotide-binding regulatory protein (G protein) is involved in functional coupling of the receptor to the Ca2+ channel. We identified the G protein involved in the stimulatory effect of TRH on the Ca2+ channel by type-selective suppression of G-protein synthesis. Antisense oligonucleotides were microinjected into GH3 cell nuclei, and 48 h after injection the TRH effect was tested. Whereas antisense oligonucleotides hybridizing to the mRNA of G(o) or Gi1 alpha-subunit sequences did not affect stimulation by TRH, oligonucleotides suppressing the expression of the Gi2 alpha subunit abolished this effect, and oligonucleotides directed against the mRNA of the Gi3 alpha subunit had less effect. The requirement of a concurrent inositol phospholipid degradation and subsequent protein kinase C (PKC) activation for the TRH effect on Ca(2+)-channel activity was demonstrated by inhibitory effects of antisense oligonucleotides directed against Gq/G11/Gz alpha-subunit sequences and treatment of GH3 cells with PKC inhibitors, respectively. Our results suggest that TRH elevates the cytosolic Ca2+ concentration in GH3 cells transiently via Ca2+ release from internal stores, followed by a phase of sustained Ca2+ influx through voltage-dependent Ca2+ channels stimulated by the concerted action of Gi2 (and Gi3) plus PKC.     

The effects of maitotoxin on 45Ca2+ flux and hormone release in GH3 rat pituitary cells

Maitotoxin has been reported to activate calcium channels and stimulate calcium-dependent functions in several tissues, but a thorough investigation of 45Ca2+ fluxes is lacking. To characterize the influence of maitotoxin on 45Ca2+ flux in greater detail, we incubated dispersed GH3 pituitary tumor cells in 45Ca2+ with maitotoxin and other agents affecting calcium channels. Within 10 sec of exposure, maitotoxin induced a net calcium influx in cells at isotopic equilibrium. Calcium uptake was concentration dependent between 0.4 and 40 ng/ml maitotoxin and was inhibited by antagonists of voltage-dependent calcium channels but not by inhibitors of sodium channels. PRL and GH release from perifused GH3 cells was stimulated within 1 min by maitotoxin. We conclude that maitotoxin causes a rapid, concentration-dependent influx of calcium through presumed voltage-dependent endogenous calcium channels, culminating in enhanced hormone release. This potent toxin may provide a more precise understanding of the role of calcium in the stimulus-secretion coupling process.     

Evidence for dual regulation by protein kinases A and C of thyrotropin-releasing hormone receptor mRNA in GH3 cells.

We showed previously that TRH down-regulates TRH receptor (TRH-R) mRNA in GH3 cells by a mechanism that appears to be mediated by protein kinase C. Here we show that vasoactive intestinal peptide (VIP) down-regulates TRH-R mRNA and present evidence that this action is mediated by protein kinase A. In GH3 cells, VIP caused a time- and concentration-dependent decrease in TRH-R mRNA. This VIP effect was simulated by 8-(4-chlorophenylthio)-cAMP, forskolin, cholera toxin and 1-methyl-3-isobutylxanthine. When cells were incubated with agents that elevate cAMP and TRH or phorbol 12-myristate 13-acetate, the decrease in TRH-R mRNA was greater than with either agent alone. When cells were pre-incubated with H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride], an inhibitor of protein kinases, the effects of VIP, TRH and phorbol 12-myristate 13-acetate were inhibited. We suggest that VIP, via protein kinase A, and TRH, via protein kinase C, dually regulate TRH-R mRNA.     

Effects of thyrotropin-releasing hormone on prolactin compartments in clonal rat pituitary tumor cells

PRL compartments were studied in a clonal strain of rat pituitary tumor cells (GH3B6). The cells were pulse-labeled for 10 min with 35S-methionine and then chased for 20 h in the absence or presence of TRH (30 nM) or cycloheximide (3.6 X 10(-5) M), or both. The specific radioactivity (SA) of PRL was followed in the cells and chase medium as a function of chase time and treatments. The transit of labeled and unlabeled PRL has been investigated in cells treated with monensin (1 microM), a drug which is known to perturb the Golgi zone. Newly synthesized PRL was rapidly (15 min of chase) and preferentially released in basal conditions. The pattern of the decay of the SA of PRL released in the medium suggested the existence of at least two PRL pools with different half-lives: 15 min and 3 h, respectively. TRH induced the preferential release of a PRL pool synthesized before the labeling pulse. Monensin decreased the basal release of total radioimmunoassayable PRL without affecting that of the newly synthesized PRL. In contrast, it did not affect the stimulating effect of TRH on the release of unlabeled PRL. These results are in favor of the existence of different intracellular routes for the basal release of PRL (mostly newly synthesized) and the TRH-stimulated release of PRL (mostly stored). Moreover, after 20 h of chase a large fraction (approximately 80%) of the labeled immunoprecipitated material remained intracellularly located and not degraded. This material was not mobilizable by TRH even in the presence of cycloheximide. Polyacrylamide gel electrophoresis analysis revealed that it consisted of large immunoreactive proteins (mol wt, 45,000 and 50,000) instead of mol wt 23,000 PRL which was found in the medium.     

Thyrotropin-releasing hormone increases phospholipase D activity through stimulation of protein kinase C in GH3 cells

Activation of phospholipase D was investigated after treatment of GH₃ cells with thyrotropin-releasing hormone. Thyrotropin-releasing hormone treatment resulted in both time- and dose-dependent increases of phospholipase D activity, translocation of protein kinase C-α and -βl isozymes from cytosol to membrane within 30 min, and approx 43-fold increase of phosphatidylinositol-specific phospholipase C activity. Intracellular calcium concentration was rapidly increased and diacyglycerol level remained high up to 3 h after the treatment. Pretreatment of the cells with U73122, a potent inhibitor of phosphatidylinositol-specific phospholipase C, inhibited thyrotropin-releasing hormone-induced phospholipase D activation. Protein kinase C activity was down-regulated by pretreatment of the GH₃ cells with either protein kinase C inhibitors (RO320432, GF109203X) or preincubation of the cells with phorbol myristrate acetate (500 nM) for 24 h. This treatment largely abolished the thyrotropin-releasing hormone-induced activation of phospholipase D, thus further confirming the involvement of protein kinase C in the activation. These results suggest that thyrotropin-releasing hormone-induced phospholipase D activation may be due to phosphatidylinositol-specific phospholipase C, and activation of protein kinase C isozymes is responsible for this stimulation.