Misfolded growth hormone causes fragmentation of the Golgi apparatus and disrupts endoplasmic reticulum-to-Golgi traffic.

In some individuals with autosomal dominant isolated growth hormone deficiency, one copy of growth hormone lacks amino acids 32-71 and is severely misfolded. We transfected COS7 cells with either wild-type human growth hormone or Delta 32-71 growth hormone and investigated subcellular localization of growth hormone and other proteins. Delta 32-71 growth hormone was retained in the endoplasmic reticulum, whereas wild-type hormone accumulated in the Golgi apparatus. When cells transfected with wild-type or Delta 32-71 growth hormone were dually stained for growth hormone and the Golgi markers beta-COP, membrin or 58K, wild-type growth hormone was colocalized with the Golgi markers, but beta-COP, membrin and 58K immunoreactivity was highly dispersed or undetectable in cells expressing Delta 32-71 growth hormone. Examination of alpha-tubulin immunostaining showed that the cytoplasmic microtubular arrangement was normal in cells expressing wild-type growth hormone, but microtubule-organizing centers were absent in nearly all cells expressing Delta 32-71 growth hormone. To determine whether Delta 32-71 growth hormone would alter trafficking of a plasma membrane protein, we cotransfected the cells with the thyrotropin-releasing hormone (TRH) receptor and either wild-type or Delta 32-71 growth hormone. Cells expressing Delta 32-71 growth hormone, unlike those expressing wild-type growth hormone, failed to show normal TRH receptor localization or binding. Expression of Delta 32-71 growth hormone also disrupted the trafficking of two secretory proteins, prolactin and secreted alkaline phosphatase. Delta 32-71 growth hormone only weakly elicited the unfolded protein response as indicated by induction of BiP mRNA. Pharmacological induction of the unfolded protein response partially prevented deletion mutant-induced Golgi fragmentation and partially restored normal TRH receptor trafficking. The ability of some misfolded proteins to block endoplasmic reticulum-to-Golgi traffic may explain their toxic effects on host cells and suggests possible strategies for therapeutic interventions.

Flavopiridol induces apoptosis and caspase-3 activation of a newly characterized Burkitt's lymphoma cell line containing mutant p53 genes.

Burkitt's lymphoma cell lines have been important in vitro models for studying the pathogenesis of Burkitt's lymphoma (BL) and for exploring new treatment strategies. A new EBV(-) Burkitt's lymphoma cell line (GA-10) was established from a patient with a clinically aggressive, chemorefractory BL and characterized. Although functional p-glycoprotein could not be demonstrated by dye-efflux assays, both p53 genes were mutated in the GA-10 cells, perhaps contributing to the resistant phenotype of the original neoplasm. Two properties of BL cells which may be useful targets for novel cytotoxic therapeutics are their surface expression of CD77, the receptor for Shiga toxin (Stx), and their high rate of proliferation. Expression of CD77 on the GA-10 cells was heterogeneous in that certain subclones expressed high levels of CD77 and correspondingly exhibited strong growth inhibition by Stx while others showed low levels of CD77 expression and weak Stx-induced growth inhibition. Flavopiridol, a potent inhibitor of cell cycle progression through G1 and G2, induced cytotoxicity of the GA-10 cells with an LC(50) of approximately 40 nM vs 70 nM for HL-60 cells (P < 0.05). The concentrations of flavopiridol at which only 10% of the cells were viable (LC(10)) were approximately 280 nM for the GA-10 cells and 520 nM for the HL-60 cells (P < 0.05). Dose-related induction of apoptosis in response to flavopiridol was demonstrated in the GA-10 cells by morphology, TUNEL assay, and activation of caspase-3. Flavopiridol was also cytotoxic to seven other BL cell lines tested. These data suggest that flavopiridol may have therapeutic value in the treatment of Burkitt's lymphoma.

Activation of MAPK by TRH requires clathrin-dependent endocytosis and PKC but not receptor interaction with beta-arrestin or receptor endocytosis.

To determine whether the interaction of the TRH receptor with beta-arrestin is necessary for TRH activation of MAPK, cells expressing either intact or truncated, internalization-defective TRH receptors were transfected with a beta-arrestin-green fluorescent protein conjugate. In cells expressing the wild-type pituitary TRH receptor, TRH caused translocation of the beta-arrestin-green fluorescent protein conjugate from the cytosol to the plasma membrane within 30 sec. After 5 min, the beta-arrestin-green fluorescent protein conjugate was visible in vesicles, where it colocalized with rhodamine-labeled TRH. In hypertonic sucrose, the beta-arrestin-green fluorescent protein conjugate translocated to the plasma membrane after TRH addition but did not internalize. In cells expressing the truncated TRH receptor, TRH did not cause translocation of the beta-arrestin-green fluorescent protein conjugate. TRH activated MAPK strongly in cells expressing intact or truncated TRH receptors, indicating that the receptor does not need to bind beta-arrestin or internalize. MAPK activation by TRH, epidermal growth factor, and phorbol ester was strongly inhibited by hypertonic sucrose and concanavalin A, which block movement of proteins into coated pits and coated pit assembly. Hypertonic sucrose did not affect MAPK activation in cells overexpressing MAPK kinase 1. Dominant negative dynamin, which blocks conversion of coated pits to vesicles, also reduced receptor internalization and TRH activation of MAPK. TRH activation of MAPK required PKC but was insensitive to pertussis toxin and did not require ras, epidermal growth factor receptor kinase, or PI3K. These results show that the TRH receptor itself does not need to bind beta-arrestin or undergo sequestration to activate MAPK but that the endocytic pathway must be intact.

Calcium responses to thyrotropin-releasing hormone, gonadotropin-releasing hormone and somatostatin in phospholipase css3 knockout mice.

These studies examined the importance of phospholipase Cbeta (PLCbeta) in the calcium responses of pituitary cells using PLCbeta3 knockout mice. Pituitary tissue from wild-type mice contained PLCbeta1 and PLCbeta3 but not PLCbeta2 or PLCbeta4. Both Galphaq/11 and Gbetagamma can activate PLCbeta3, whereas only Galphaq/11 activates PLCss1 effectively. In knockout mice, PLCbeta3 was absent, PLCbeta1 was not up-regulated, and PLCbeta2 and PLCbeta4 were not expressed. Since somatostatin inhibited influx of extracellular calcium in pituitary cells from wild-type and PLCbeta3 knockout mice, the somatostatin signal pathway was intact. However, somatostatin failed to increase intracellular calcium in pituitary cells from either wild-type or knockout mice under a variety of conditions, indicating that it did not stimulate PLCbeta3. In contrast, somatostatin increased intracellular calcium in aortic smooth muscle cells from wild-type mice, although it evoked no calcium response in cells from PLCbeta3 knockout animals These results show that somatostatin, like other Gi/Go-linked hormones, can stimulate a calcium transient by activating PLCbeta3 through Gbetagamma, but this response does not normally occur in pituitary cells. The densities of Gi and Go, as well as the relative concentrations of PLCbeta1 and PLCbeta3, were similar in cells that responded to somatostatin with an increase in calcium and pituitary cells. Calcium responses to 1 nM and 1 microM TRH and GnRH were identical in pituitary cells from wild-type and PLCbeta3 knockout mice, as were responses to other Gq-linked agonists. These results show that in pituitary cells, PLCbeta1 is sufficient to transmit signals from Gq-coupled hormones, whereas PLCbeta3 is required for the calcium-mobilizing actions of somatostatin observed in smooth muscle cells.

Rapid turnover of calcium in the endoplasmic reticulum during signaling. Studies with cameleon calcium indicators.

HEK293 cells expressing the thyrotropin-releasing hormone (TRH) receptor were transfected with cameleon Ca(2+) indicators designed to measure the free Ca(2+) concentration in the cytoplasm, [Ca(2+)](cyt), and the endoplasmic reticulum (ER), [Ca(2+)](er). Basal [Ca(2+)](cyt) was about 50 nm; thyrotropin-releasing hormone (TRH) or other agonists increased [Ca(2+)](cyt) to 1 micrometer or higher. Basal [Ca(2+)](er) averaged 500 micrometer and fell to 50-100 micrometer over 10 min in the presence of thapsigargin. TRH consistently decreased [Ca(2+)](er) to 100 micrometer, independent of extracellular Ca(2+), whereas agonists for endogenous receptors generally caused a smaller decline. When added with thapsigargin, all agonists rapidly decreased [Ca(2+)](er) to 5-10 micrometer, indicating that there is substantial store refilling during signaling. TRH increased [Ca(2+)](cyt) and decreased [Ca(2+)](er) if applied after other agonists, whereas other agonists did not alter [Ca(2+)](cyt) or [Ca(2+)](er) if added after TRH. When Ca(2+) was added back to cells that had been incubated with TRH in Ca(2+)-free medium, [Ca(2+)](cyt) and [Ca(2+)](er) increased rapidly. The increase in [Ca(2+)](er) was only partially blocked by thapsigargin but was completely blocked if cells were loaded with 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. In conclusion, these new Ca(2+) indicators showed that basal [Ca(2+)](er) is approximately 500 micrometer, that [Ca(2+)](er) has to be >100 micrometer to support an increase in [Ca(2+)](cyt) by agonists, and that during signaling, intracellular Ca(2+) stores are continuously refilled with cytoplasmic Ca(2+) by the sarcoendoplasmic reticulum Ca(2+)-ATPase pump.

Genetic alteration of phospholipase C beta3 expression modulates behavioral and cellular responses to mu opioids.

Morphine and other micro opioids regulate a number of intracellular signaling pathways, including the one mediated by phospholipase C (PLC). By studying PLC beta3-deficient mice, we have established a strong link between PLC and mu opioid-mediated responses at both the behavioral and cellular levels. Mice lacking PLC beta3, when compared with the wild type, exhibited up to a 10-fold decrease in the ED(50) value for morphine in producing antinociception. The reduced ED(50) value was unlikely a result of changes in opioid receptor number or affinity because no differences were found in whole-brain B(max) and K(d) values for mu, kappa, and delta opioid receptors between wild-type and PLC beta3-null mice. We also found that opioid regulation of voltage-sensitive Ca(2+) channels in primary sensory neurons (dorsal root ganglion) was different between the two genotypes. Consistent with the behavioral findings, the specific mu agonist [D-Ala(2),(Me)Phe(4),Gly(ol)(5)]enkephalin (DAMGO) induced a greater whole-cell current reduction in a greater proportion of neurons isolated from the PLC beta3-null mice than from the wild type. In addition, reconstitution of recombinant PLC protein back into PLC beta3-deficient dorsal root ganglion neurons reduced DAMGO responses to those of wild-type neurons. In neurons of both genotypes, activation of protein kinase C with phorbol esters markedly reduced DAMGO-mediated Ca(2+) current reduction. These data demonstrate that PLC beta3 constitutes a significant pathway involved in negative modulation of mu opioid responses, perhaps via protein kinase C, and suggests the possibility that differences in opioid sensitivity among individuals could be, in part, because of genetic factors.

Signal transduction and hormone-dependent internalization of the thyrotropin-releasing hormone receptor in cells lacking Gq and G11.

The thyrotropin-releasing hormone (TRH) receptor was expressed in embryonic fibroblasts from mice lacking the alpha subunits of Gq and G11 (Fq/11 cells) to determine whether G protein coupling is necessary for agonist-dependent receptor internalization. Neither TRH nor agonists acting on endogenous receptors increased intracellular calcium unless the cells were co-transfected with the alpha subunit of Gq. In contrast, temperature-dependent internalization of [3H]MeTRH in Fq/11 cells was the same whether Gqalpha was expressed or not. A rhodamine-labeled TRH analog and fluorescein-labeled transferrin co-localized in endocytic vesicles in Fq/11 cells, indicating that endocytosis took place via the normal clathrin pathway. Cotransfection with beta-arrestin or V53D beta-arrestin increased TRH-dependent receptor sequestration. Fq/11 cells were co-transfected with the TRH receptor and a green fluorescent protein (GFP)-beta-arrestin conjugate. GFP-beta-arrestin was uniformly distributed in the cytoplasm of untreated cells and quickly translocated to the periphery of the cells when TRH was added. A truncated TRH receptor that lacks potential phosphorylation sites in the cytoplasmic carboxyl terminus signaled but did not internalize or cause membrane localization of GFP-beta-arrestin. These results prove that calcium signaling by the TRH receptor requires coupling to a G protein in the Gq family, but TRH-dependent binding of beta-arrestin and sequestration do not.

Expression cloning and characterization of PREB (prolactin regulatory element binding), a novel WD motif DNA-binding protein with a capacity to regulate prolactin promoter activity.

Previous studies have implied that a transcription factor(s) other than Pit-1 is involved in homeostatic regulation of PRL promoter activity via Pit-1-binding elements. One such element, 1P, was employed to clone from a rat pituitary cDNA expression library a novel 417-amino acid WD protein, designated PREB (PRL regulatory element binding) protein. PREB contains two PQ-rich potential transactivation domains, but no apparent DNA-binding motif, and exhibits sequence-specific binding to site 1P, to a site nonidentical to that for Pit-1. The PREB gene (or a related gene) is conserved, as an apparently single copy, in rat, human, fly, and yeast. A single approximately 1.9-kb PREB transcript accumulates in GH3 rat pituitary cells, to levels similar to Pit-1 mRNA. PREB transcripts were detected in all human tissues examined, but the observation of tissue-specific multiple transcript patterns suggests the possibility of tissue-specific alternative splicing. RT-PCR analysis of human brain tumor RNA samples suggested region-specific expression of PREB transcripts in brain. Western and immunocytochemical analysis implied that PREB accumulates specifically in GH3 cell nuclei. Transient transfection employing PREB-negative C6 rat glial cells showed that PREB is as active as, and additive with, Pit-1 in transactivation of a PRL promoter construct, and that PREB, but not Pit-1, can mediate transcriptional activation by protein kinase A (PKA). Expression in GH3 cells of a GAL4-PREB fusion protein both strongly transactivated a 5XGAL indicator construct and yielded a further stimulation of expression of this construct by coexpressed PKA, implying that PREB can mediate both basal and PKA-stimulated transcriptional responses in pituitary cells. These observations imply that PREB will prove to play a significant transcriptional regulatory role, both in the pituitary and in other organs in which transcripts of its gene are expressed.

Receptors for thyrotropin-releasing hormone on rat lactotropes and thyrotropes.

Primary cultures of rat pituitary cells were stained with an antibody to the native thyrotropin-releasing hormone (TRH) receptor and with a bioactive, fluorescent analogue of TRH, Rhod-TRH. Rhod-TRH specifically stained 86% of lactotropes and 21% of nonlactotropes from primary pituitary cell cultures. Lactotropes and thyrotropes accounted for 90% of cells that stained with Rhod-TRH, but there were occasional lactotropes and thyrotropes that did not show detectable staining with antireceptor antibodies or with Rhod-TRH. The intensity of staining was generally higher in the GH3 line of tumor cells than in normal pituicytes, and 100% of the tumor cells stained with Rhod-TRH. To determine whether the TRH receptor undergoes ligand-directed endocytosis in normal cells, TRH receptor immunocytochemistry was performed before and after TRH binding. TRH receptors were localized on the surface of cells prior to TRH exposure, and Rhod-TRH fluorescence was confined to the plasma membrane when TRH binding was performed at 0 degrees C, where endocytosis is blocked. When cells were incubated with TRH at 37 degrees C, receptors were found in intracellular vesicles in both lactotropes and thyrotropes, and Rhod-TRH was rapidly internalized into endosomes at elevated temperatures. Internalization of Rhod-TRH was inhibited by hypertonic sucrose, indicating that it occurs through clathrin-coated pits. These findings show that some of the heterogeneity in the secretory and calcium responses of pituicytes to TRH occurs at the level of the TRH receptor.

Fluorescence methods to assess multidrug resistance in individual cells.

PURPOSE: Microscopic methods to measure the activity of drug extrusion systems important in multidrug resistance in individual cells were developed. METHODS: Multidrug-resistant (MDR) and parental lines of hamster CHO and pituitary GH3 cells were incubated with the acetoxymethylester (AM) forms of several fluorescent calcium-sensing dyes, fura2, indo1 and fluo3. The AM forms of these compounds are hydrolyzed by intracellular esterases and then trapped in cells, and the AM forms of the dyes are excellent substrates for P-glycoprotein (Pgp). RESULTS: The fluorescent free acid forms of fura2, indol and fluo3 did not accumulate in MDR lines unless a chemosensitizer such as cyclosporin A, R(+)verapamil, quinidine, or progesterone was included during loading to prevent the cells from extruding the AM forms of the dyes before they could be hydrolyzed. Cyclosporin A increased the fluorescence due to intracellularly trapped fura2 free acid from 8- to 20-fold and was maximally effective at < 5 microM. Fluorescence microscopy was employed to measure fura2 free acid accumulation by parental and MDR cell lines using excitation at the Ca2+-insensitive wavelength. When MDR cells were incubated with rhodamine 123 and fura2/AM, no fluorescence was detectable. Cellular fluorescence was dramatically increased by inclusion of cyclosporin A, quinidine, progesterone, or R(+)verapamil. There was no measurable decline in the fura2 free acid fluorescence in 1 h while the fluorescence due to rhodamine 123 diminished rapidly in cells overexpressing Pgp. CONCLUSIONS: These fluorescence methods detect drug-extruding activity in individual cells and therefore have the potential to provide complementary information to studies quantifying protein or mRNA levels of Pgp or other efflux pumps. In addition, they provide a rapid and quantifiable method for screening multidrug resistance reversing agents.

Signal transduction, desensitization, and recovery of responses to thyrotropin-releasing hormone after inhibition of receptor internalization.

Three independent methods were used to block internalization of the TRH receptor: cells were infected with vaccinia virus encoding a dominant negative dynamin, incubated in hypertonic sucrose, or stably transfected with a receptor lacking the C-terminal tail. Internalization was blocked in all three paradigms as judged by microscopy using a fluorescently labeled TRH agonist and biochemically. The initial inositol trisphosphate (IP3) and Ca2+ responses to TRH were normal when internalization was inhibited. The IP3 increase was sustained rather than transient, however, in cells expressing the truncated TRH receptor, implying that the C-terminal tail of the receptor may be important for uncoupling from phospholipase C. After withdrawal of TRH, cells were refractory to TRH until both ligand dissociation and resensitization of the receptor had occurred. When surface-bound TRH was removed by a mild acid wash, which did not impair receptor function, neither wild-type nor truncated receptors were able to generate full IP3 responses for about 10 min. The rate of recovery was not altered by blocking internalization. Recovery of intracellular Ca2+ responses also depended on the rate of Ca2+ pool refilling. In summary, in the continued presence of TRH, phospholipase C activity declines quickly due to receptor uncoupling; this desensitization does not take place for the truncated receptor. After TRH is withdrawn, cells are refractory to TRH. Before cells can respond, TRH must dissociate and a resensitization step, which takes place on the plasma membrane and does not require the C-terminal tail of the receptor, must occur.

Desensitization of thyrotropin-releasing hormone receptor-mediated responses involves multiple steps.

Desensitization and recovery of the inositol 1,4,5-trisphosphate (IP3) and intracellular free calcium concentration ([Ca2+]i) responses to thyrotropin-releasing hormone (TRH) were measured in HEK293 cells stably expressing the G protein-coupled TRH receptor. TRH caused a large, rapid, and transient increase in IP3 and a biphasic increase in [Ca2+]i. Desensitization of the TRH response was measured by exposing cells to TRH, washing, and then incubating the cells in hormone-free medium before reintroducing TRH and measuring IP3, [Ca2+]i, and intracellular Ca2+ pool size. When cells were incubated with 1 microM TRH for 10 s or 10 min and reexposed to TRH, there was almost no IP3 or [Ca2+]i increase. The IP3 response recovered first, followed by the [Ca2+]i response. The ionomycin-releasable intracellular Ca2+ pool was almost completely depleted by TRH, and pool refilling was slow. Thrombin, endothelin, and carbachol, when combined, stimulated large increases in IP3 and [Ca2+]i, but did not block the IP3 or [Ca2+]i responses to TRH measured 10 min later. In contrast, cells exposed to TRH first responded to combined agonists with a nearly normal increase in IP3, but no rise in [Ca2+]i. Thus, the IP3 response to TRH displays homologous desensitization, whereas the [Ca2+]i response displays heterologous desensitization because depletion of intracellular Ca2+ pools prevents responses to other hormones.

Lead uptake in brain capillary endothelial cells: activation by calcium store depletion.

The mechanism by which lead crosses the blood-brain barrier is not known. Brain capillary endothelial cells, which form tight junctions with each other, are an important component of the blood-brain barrier. Lead must traverse these cells to reach the brain. In the present study, uptake of lead was followed in primary cultures of bovine brain capillary endothelial cells. Lead uptake into cells was measured by monitoring the fluorescence of cells loaded with indo-1 at a wavelength where indo-1 fluorescence is independent of calcium but quenched by binding of lead. Lead uptake was visualized with digital images recorded with a fluorescence microscope. Lead added to the extracellular medium caused fluorescence quench over time which was reversed upon addition of a membrane permeant heavy metal chelator. Lead uptake by cells in suspension, measured by fluorescence spectroscopy, exhibited time and concentration dependence. Lead uptake was enhanced following depletion of intracellular Ca2+ stores by the addition of thapsigargin, cyclopiazonic acid, or tert-butylhydroquinone, inhibitors of the sarco/endoplasmic reticulum calcium ATPase. SK&F 96365, which blocks store-operated calcium channels, inhibited the stimulation of lead uptake by thapsigargin. These results indicate that indo-1 fluorescence quench is a useful method for investigation of lead uptake in brain capillary endothelial cells. Furthermore, entry of lead into these cells is activated by the depletion of intracellular Ca2+ stores and may occur via store-operated cation channels.

Detection in living cells of Ca2+-dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. A new class of fluorescent indicators.

We have designed a novel fluorescent indicator composed of two green fluorescent protein variants joined by the calmodulin-binding domain from smooth muscle myosin light chain kinase. When (Ca2+)4-calmodulin is bound to the indicator (Kd = 0.4 nM), fluorescence resonance energy transfer between the two fluorophores is attenuated; the ratio of the fluorescence intensity measured at 505 nm to the intensity measured at 440 nm decreases 6-fold. Images of microinjected living cells demonstrate that emission ratios can be used to monitor spatio-temporal changes in the fluorescence of the indicator. Changes in indicator fluorescence in these cells are coupled with no discernible lag (<1 s) to changes in the cytosolic free Ca2+ ion concentration, ranging from below 50 nM to approximately 1 microM. This observation suggests that the activity of a calmodulin target with a typical 1 nM affinity for (Ca2+)4-calmodulin is responsive to changes in the intracellular Ca2+ concentration over the physiological range. It is likely that the indicator we describe can be modified to detect the levels of ligands and proteins in the cell other than calmodulin.

Effect of cell type on the subcellular localization of the thyrotropin-releasing hormone receptor.

The localization of an epitope-tagged receptor for thyrotropin-releasing hormone (TRH) expressed in different cell contexts was studied with immunofluorescence microscopy. In pituitary lactotrophs, which normally express TRH receptors, and in AtT20 pituitary corticotrophs, TRH receptor immunoreactivity was primarily confined to the plasma membrane. In HEK 293 and COS7 cells, TRH receptors were predominantly intracellular. In transiently transfected COS7 cells, the TRH receptor colocalized with endoplasmic reticulum and Golgi markers. The pattern of TRH receptor immunofluorescence was the same over a wide range of receptor expression in transiently transfected COS7 cells, and all cell lines bound similar amounts of 3H- and rhodamine-labeled TRH analogs, suggesting that cell-specific differences in TRH receptor localization were not simply the result of overexpression. In all cell contexts, TRH receptors on the plasma membrane underwent extensive ligand-driven endocytosis. Inhibitors of glycosylation did not alter the subcellular distribution of receptors. In HEK 293 cells expressing the transfected TRH receptor, protein synthesis inhibitors caused translocation of intracellular receptors to the cell surface, as shown by a marked increase in cell surface immunofluorescence and [3H][N3-methyl-His2]TRH binding. These results demonstrate that the subcellular localization of the TRH receptor depends on the cell context in which it is expressed and that intracellular receptors are capable of translocation to the plasma membrane.

Cellular uptake of lead is activated by depletion of intracellular calcium stores.

The mechanisms of cellular lead uptake were characterized using a fluorescence method in cells loaded with indo-1. Pb2+ bound to intracellular indo-1 with much higher affinity than Ca2+ and quenched fluorescence at all wavelengths. Pb2+ uptake into pituitary GH3 cells, glial C6 cells, and a subclone of HEK293 cells was assessed by fluorescence quench at a Ca2+-insensitive emission wavelength. Pb2+ uptake was concentration- and time-dependent. Pb2+ uptake in all three cell types occurred at a much faster rate when intracellular Ca2+ stores were depleted by two different methods: addition of drugs that inhibit the endoplasmic reticulum Ca2+ pump (thapsigargin, cyclopiazonic acid, and tert-butylhydroquinone), and prolonged incubation of cells in Ca2+-free media. Application of receptor agonists, which deplete intracellular Ca2+ stores via inositol trisphosphate-sensitive channels, did not activate Pb2+ uptake. Agonists were just as effective as thapsigargin in stimulating uptake of Ca2+ but less so in stimulating uptake of Mn2+. Basal and stimulated Pb2+ uptake were partially reduced by 1 mM extracellular Ca2+ and strongly inhibited by 10 mM Ca2+. Pb2+ entry in GH3 cells was inhibited by two drugs that block capacitative Ca2+ entry, La3+ and SK&F 96365. Depolarization of electrically excitable GH3 cells increased the initial rate of Pb2+ uptake 1.6-fold, whereas thapsigargin increased uptake 12-fold. In conclusion, Pb2+ crosses the plasma membrane of GH3, C6, and HEK293 cells via channels that are activated by profound depletion of intracellular Ca2+ stores.

Characterization of the calcium response to thyrotropin-releasing hormone in lactotrophs and GH cells.

Thyrotropin-releasing hormone (TRH) acts via a G-protein-coupled receptor on lactotrophs to increase the intracellular free calcium ion concentration, [Ca(2+)](i). The [Ca(2+)](i) response depends on both TRH concentration and the duration of TRH exposure. An initial, short-lived [Ca(2+)](i) spike results from release of Ca(2+) from intracellular stores, whereas a later sustained [Ca(2+)](i) increase, often characterized by [Ca(2+)](i) oscillations, results from an influx of extracellular Ca(2+) through both voltage-gated and non-voltage-gated, store-operated Ca(2+) channels. The initial spike phase predominates at high doses of TRH, whereas the plateau phase predominates at low doses. The mechanisms underlying the complex [Ca(2+)](i) response to TRH are discussed.

Thyrotropin-releasing hormone-induced intracellular calcium responses in individual rat lactotrophs and thyrotrophs.

Calcium responses to TRH were recorded for individual cells cultured from rat anterior pituitary tissue loaded with fura-2, and cell type was subsequently identified by immunocytochemistry. At 100 nM and 1 microM, TRH stimulated a single transient spike of intracellular free calcium ([Ca2+]i) in 95-100% of lactotrophs. At a concentration of 10 nM or less, the proportion of TRH-responsive cells decreased, and the [Ca2+]i responses became more heterogeneous, consisting of a biphasic response in which an initial [Ca2+]i spike was followed by a sustained elevation of [Ca2+]i or [Ca2+]i oscillations. Initiation of TRH-induced oscillations required the release of intracellular Ca2+ from thapsigargin-sensitive stores, whereas maintenance of the oscillations required influx of extracellular Ca2+ through nimodipine-sensitive Ca2+ channels. The amplitude of the initial [Ca2+]i rise increased from 0.1-10 nM TRH and was not significantly reduced by removal of extracellular Ca2+. The duration of the initial [Ca2+]i transient was significantly shorter at 1 microM than at 1 nM TRH. When TRH was added to cells that had been treated with thapsigargin to block the agonist-induced [Ca2+]i increase, TRH often decreased [Ca2+]i, particularly in cells with high [Ca2+]i. These results suggest that TRH and elevated [Ca2+]i act as coactivators of Ca2+ efflux, which helps terminate the agonist-evoked [Ca2+]i transient. In addition, TRH caused increases in [Ca2+]i in individual rat thyrotrophs, and these responses were heterogeneous. TRH stimulated a [Ca2+]i response in a lesser proportion of thyrotrophs from euthyroid compared to hypothyroid male rats. Essentially all TRH-responsive cells stained for either PRL or TSH.

Thyrotropin (TSH)-releasing hormone stimulates TSH beta promoter activity by two distinct mechanisms involving calcium influx through L type Ca2+ channels and protein kinase C.

TRH stimulates rat (r) TSH beta gene promoter activity at two distinct response elements, which also respond to protein kinase C-signaling pathways. The dependence of TRH-stimulated transcription of the TSH beta gene on a rise in intracellular calcium [Ca2+]i, and on the necessity for Ca2+ influx through L-type voltage-gated calcium channels was investigated in two transfected cell lines and in normal thyrotropes. The transcription rate of the homologous gene in normal thyrotropes was measured by nuclear run-off assays. Bay K8644, an L channel agonist, stimulated TSH beta gene transcription 6-fold, and TRH stimulation of TSH beta gene transcription was partially blocked by nimodipine, an L channel antagonist, while phorbol 12-myristate-13-acetate (PMA)-stimulated transcription was not. Bay K8644 plus TRH had a greater effect than either treatment alone. Constructs of the 5'-flanking region of the TSH beta gene fused to the luciferase reporter (TSH beta LUC) were then transfected into excitable GH3 pituitary cells. TSH beta LUC was stimulated 2- to 5-fold by 1 nM TRH or 100 nM Bay K8644, and the TRH effect was nearly abolished by nimodipine or chelation of external Ca2+. Constructs containing isolated TRH-responsive elements fused to a heterologous promoter responded similarly. The protein kinase C activator, PMA (100 nM) also stimulated TSH beta LUC transcription, but its effect was not inhibited by nimodipine. A stable heterologous cell line containing the mouse TRH receptor was constructed by transfection of nonexcitable 293 cells, which lack L channel activity. In the resultant 301 cells, TSH beta LUC activity was increased 2- to 3-fold by TRH or PMA; nimodipine, Bay K8644, and removal of extracellular Ca2+ had no effect. We conclude that TRH stimulation of TSH beta gene transcription requires Ca2+ release from inositol triphosphate-sensitive stores and Ca2+ influx via L-type calcium channels in GH3 cells, but in transfected 293 cells TRH activation of protein kinase C plays a predominant role in activating TSH beta. Both mechanisms appear to be operative in normal thyrotropes.