Rat brain TRH receptors: kinetics, pharmacology, distribution and ionic effects

Optimal conditions for measuring receptor binding for thyrotropin-releasing hormone (TRH) in the rat central nervous system (CNS) have been determined using 3H-labelled [3-Me-His2]TRH [( 3H]MeTRH). Binding assays conducted at 0 degree C for 5-6 h using sodium phosphate- and/or Hepes-buffered tissue resuspensions, with subsequent filtration through Whatman GF/B filters, yielded the best results. Association and dissociation of [3H]MeTRH binding to amygdala membranes were time and temperature dependent. Dissociation kinetics appeared biphasic. Progressive reduction in receptor affinity and capacity and increased radioligand breakdown were observed at elevated temperatures. Bacitracin (25-1000 microM) prevented peptide degradation but inhibited receptor binding (8-37%). Detailed competition experiments using MeTRH and other drugs yielded a pharmacological profile similar to that observed previously in other tissues indicating TRH receptor identification. Highest density of TRH receptors was observed in the retina and numerous limbic areas. Monovalent and divalent cations modulated [3H]MeTRH binding by reducing apparent receptor number.     

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.     

Solubilization of receptors for thyrotropin-releasing hormone from GH4C1 rat pituitary cells: demonstration of guanyl nucleotide sensitivity

TRH receptors have been solubilized from GH4C1 cells using the plant glycoside digitonin. Solubilized receptors retain the principal binding characteristics exhibited by the TRH receptor in intact pituitary cells and their membranes. The binding of the methylhistidyl derivative of TRH [( 3H]MeTRH) attained equilibrium within 2-3 h at 4 C, and it was reversible, dissociating with a t1/2 of 7 h. Analysis of [3H]MeTRH binding to soluble receptors at 4 C yielded a dissociation constant (Kd) of 3.8 nM and a total binding capacity (Bmax) of 3.9 pmol/mg protein. Peptides known to interact with non-TRH receptors on GH cells failed to interfere with the binding of [3H]MeTRH, indicating that the TRH binding was specific. Chlordiazepoxide, a competitive antagonist for TRH action in GH cells, inhibited TRH binding to soluble receptors with an IC50 of 11 microM. When [3H]MeTRH was bound to membranes and the membrane proteins were then solubilized, we found enhanced dissociation of the prebound [3H]MeTRH from its solubilized receptor by guanyl nucleotides. Maximal enhancement of [3H]MeTRH dissociation by 10 microM GTP gamma S occurred within about 45 min at 22 C. GTP gamma S, GTP, GDP beta S, and GDP were all effectors of [3H]MeTRH dissociation, exhibiting EC50s in the range of 14-450 nM. The rank order of potency of the tested nucleotides was GTP gamma S greater than GTP congruent to GDP beta S greater than GDP much greater than ATP gamma S greater than GMP. We conclude that TRH receptors have been solubilized from GH cells with digitonin and retain the binding characteristics of TRH receptors in intact pituitary cells. Furthermore, prebinding [3H]MeTRH to GH4C1 cell membranes results in the solubilization of a complex in which the TRH receptor is linked functionally to a GTP binding protein.     

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.     

Beta-arrestin mediates desensitization and internalization but does not affect dephosphorylation of the thyrotropin-releasing hormone receptor

The G protein-coupled thyrotropin-releasing hormone (TRH) receptor is phosphorylated and binds to beta-arrestin after agonist exposure. To define the importance of receptor phosphorylation and beta-arrestin binding in desensitization, and to determine whether beta-arrestin binding and receptor endocytosis are required for receptor dephosphorylation, we expressed TRH receptors in fibroblasts from mice lacking beta-arrestin-1 and/or beta-arrestin-2. Apparent affinity for [(3)H]MeTRH was increased 8-fold in cells expressing beta-arrestins, including a beta-arrestin mutant that did not permit receptor internalization. TRH caused extensive receptor endocytosis in the presence of beta-arrestins, but receptors remained primarily on the plasma membrane without beta-arrestin. beta-Arrestins strongly inhibited inositol 1,4,5-trisphosphate production within 10 s. At 30 min, endogenous beta-arrestins reduced TRH-stimulated inositol phosphate production by 48% (beta-arrestin-1), 71% (beta-arrestin-2), and 84% (beta-arrestins-1 and -2). In contrast, receptor phosphorylation, detected by the mobility shift of deglycosylated receptor, was unaffected by beta-arrestins. Receptors were fully phosphorylated within 15 s of TRH addition. Receptor dephosphorylation was identical with or without beta-arrestins and almost complete 20 min after TRH withdrawal. Blocking endocytosis with hypertonic sucrose did not alter the rate of receptor phosphorylation or dephosphorylation. Expressing receptors in cells lacking Galpha(q) and Galpha(11) or inhibiting protein kinase C pharmacologically did not prevent receptor phosphorylation or dephosphorylation. Overexpression of dominant negative G protein-coupled receptor kinase-2 (GRK2), however, retarded receptor phosphorylation. Receptor activation caused translocation of endogenous GRK2 to the plasma membrane. The results show conclusively that receptor dephosphorylation can take place on the plasma membrane and that beta-arrestin binding is critical for desensitization and internalization.     

Modulation of Receptors for Thyrotropin-Releasing Hormone by Benzodiazepines: Brain Regional Differences

The benzodiazepines (BZDs) chlordiazepoxide (CDE), diazepam (DZM), and flurazepam (FLM) inhibited receptor binding for thyrotropin-releasing hormone (TRH) with low micromolar potency. In contrast, numerous other categories of drugs were previously shown to be inactive. Scatchard analysis of competition data suggested that the BZDs reduced TRH receptor affinity, consistent with competitive inhibition. Receptors from amygdala, retina, and pituitary appeared more sensitive to inhibition by BZDs than those from hypothalamus, hippocampus, spinal cord, or cerebellum. The latter four regions also gave shallower inhibition curves. CDE revealed an apparently biphasic dissociation of [3-Me-His2]TRH([3H]MeTRH) from amygdala membranes at 4 degrees C, with kinetics similar to those with TRH. These results suggest that TRH receptors in the brain are heterogeneous and that certain BZDs in high therapeutic concentrations may exert central effects through actions at TRH receptors or coupled proteins.     

Phosphorylation of the epidermal growth factor receptor at threonine 654 inhibits ligand-induced internalization and down-regulation

To assess the functional significance of phosphorylation of the epidermal growth factor (EGF) receptor at Thr654, we compared the effects of 12-O-tetradecanoyl-13-acetate (TPA) on ligand-induced internalization and down-regulation between wild-type and mutant receptors that contain an alanine substitution at position 654. Activation of protein kinase C with TPA blocked EGF-induced internalization and down-regulation of Thr654 receptors and inhibited in vivo tyrosine kinase activity by 80%. TPA did not inhibit transferrin receptor internalization or constitutive EGF receptor internalization, suggesting that protein kinase C activation inhibits only the ligand-induced process. Inhibition by TPA of induced internalization, down-regulation, and kinase activity required threonine at position 654 since full-length Ala654 EGF receptors were significantly resistant to TPA inhibition of these ligand-induced activities. However, C'-terminal truncation further enhanced this resistance to TPA inhibition. The EGF-dependent internalization of kinase-inactive receptors truncated at residue 1022 was also impaired by TPA in Thr654 receptors, but not in Ala654 receptors, indicating that phosphorylation at Thr654 also interferes with tyrosine kinase-independent receptor activities. We conclude that the dominant regulatory effect of protein kinase C on the EGF receptor is mediated through phosphorylation at Thr654 which effectively inactivates the receptor. The submembrane region of the EGF receptor appears to regulate transmission of conformational information from the extracellular ligand-binding site to the cytoplasmic kinase and regulatory domains.     

Signals for activation and proliferation of murine T lymphocyte clones

Biochemical signals required for the growth of T cell clones were studied. Antigen-specific helper T cell clones, 6-1 and KO.6, could enter the state similar to the resting state where the cells expressed only small numbers of interleukin 2 (IL2) receptors and could not respond to IL2 without antigenic stimulation. A combination of a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), and a calcium ionophore, A23187, induced the expression of IL2 receptors on resting 6-1 cells and induced DNA synthesis in the presence of IL2. TPA alone did not induce IL2 receptors. A23187 induced the expression of the receptors to some extent but did not induce DNA synthesis even in the presence of IL2. IL2 receptors induced by A23187 alone were mostly low affinity receptors, whereas the combination of TPA and A23187 induced high affinity receptors in addition to low affinity receptors. Resting KO.6 cells produced IL2 in response to a combination of TPA and A23187, whereas either one of the agents did not induce the production of IL2. Dicaprylin, a permeable diacylglycerol and a potent activator of protein kinase C (the Ca2+/phospholipid-dependent enzyme) could replace TPA in both cases when dicaprylin was repeatedly added to the culture. These results suggest that strong and continuous activation of protein kinase C together with calcium mobilization is required for IL2 production and IL2 receptor expression. On the contrary, signals for DNA synthesis generated by binding of IL2 to IL2 receptors are different from those for IL2 production and IL2 receptor expression, as the combination of TPA and A23187 could not induce DNA synthesis without IL2.     

Thyrotropin-releasing hormone binding to the mouse pituitary receptor does not involve ionic interactions. A model for neutral peptide binding to G protein-coupled receptors.

Thyrotropin-releasing hormone, TRH (< Glu-His-Proamide), and [N tau-Me-His]TRH (MeTRH) are present as neutral and positively charged forms at physiologic pH, and it was possible that they bind to the TRH receptor (TRH-R) as charged (protonated) species. Binding affinities of TRH and MeTRH to endogenous rat TRH-Rs and to transfected wild type mouse TRH-Rs decreased below pH 7.1. Half-maximal decreases in binding occurred at the approximate pK alpha values of these ligands. Asp to Ala mutations in extracellular loop 1, TM-4, and TM-5 did not decrease binding affinity, but an Asp to Ala mutation in TM-2 caused the affinity to decrease 8-fold. The pH dependences of binding of MeTRH, however, were similar in wild type and all mutant receptors and were consistent with the protonated form of MeTRH binding less well. Thus, the binding of TRH to its receptor does not involve ionic interactions and may be a prototype for binding of neutral peptide ligands to G protein-coupled receptors.     

A novel substance P binding site in rat brain regions modulates TRH receptor binding

Binding sites for thyrotropin-releasing hormone (TRH) were labelled with [³H](2-Me-His³)TRH ([³H]MeTRH) on membranes from rat brain regions at 0°C for 5 h. Amygdaloid membranes bound [³H]MeTRH with high-affinity (K _d=3.1±0.5 nM (n=4)). Five TRH analogs competed for this binding with the same rank order and with affinities that matched the pharmacological specificity of pituitary TRH receptors. Substance P (SP) and its C-terminal fragments reduced amygdaloid TRH receptor binding in a concentration dependent manner (IC₅₀ for SP=65 M). The rank order of potency of SP analogs at inhibiting TRH receptor binding was: SP>nonapeptide (3–11)>hexapeptide (6–11)>heptapeptide (5–11)>pentapeptide (7–11). However, other tachykinins were inactive in this system. SP was a potent inhibitor of [³H]MeTRH binding in hippocampus> spinal cord>retina>n. accumbens>hypothalamus>amygdaloid>olfactory bulb pituitary>pons/medulla in parallel assays. In amygdaloid membranes SP (50 M) reduced the apparent maximum receptor density by 39% (p<0.01) without altering the binding affinity, and 100 M SP induced a biphasic dissociation of [³H]MeTRH with kinetics faster than those induced by both TRH (10 M) and serotonin (100 M). In contrast, other neuropeptides such as neurotensin, proctolin, angiotensin II, bombesin and luteinizing hormone releasing hormone did not significantly inhibit [³H]MeTRH binding to amydaloid membranes. Thus, the SP site with low affinity in the rat brain is not like any of the previously described tachykinin/neurokinin binding sites but resembles the site found on neuroblastoma cells (108CC15) and on adrenal chromaffin cells that modulate cation permeability and nicotinic receptors respectively. The physiological role of these atypical SP sites in the rat brain remains to be determined.A preliminary account of these studies has been presented to the British Pharmacological Society (9).     

Protein kinase C mediated regulation of calcium channels in PC-12 pheochromocytoma cells

Depolarization of PC-12 pheochromocytoma cells with K+ produces an immediate increase in catecholamine release. The stimulation of release is blocked by Co2+, removal of extracellular Ca2+ or by dihydropyridine drugs such as nitrendipine. Release is enhanced by other dihydropyridines such as BAY K8644. Release is accompanied by a voltage dependent uptake of 45Ca2+ which is also blocked by Co2+ or nitrendipine and enhanced by BAY K8644. The phorbol ester phorbol 12-myristate-13-acetate (TPA) in the range 10(-9)-10(-6) M produced little effect by itself but augmented the K+ evoked release of catecholamine. An analog of TPA which does not activate protein kinase C was ineffective. In contrast, TPA in the same concentration range blocked influx of 45Ca2+ induced by 70 mM K+ or 70 mM K+/BAY K8644. 45Ca2+ influx produced by A23187 was not blocked by TPA. The results suggest a system by which protein kinase C may regulate the output of transmitters from secretory cells.     

In vitro secretion of calcitonin from a rat C cell line: effect of repetitive stimulation with the calcium channel agonist BAY K 8644.

Calcium and BAY K 8644 acutely stimulate calcitonin secretion by influx of extracellular calcium (Ca) through voltage-dependent calcium channels, leading to an increase in cytosolic free Ca. Repetitive exposure to BAY K 8644 (10(-6) M) resulted in an increase in calcitonin (CT) secretion in the rat C-cell line (rMTC 6-23) lasting 9 hours, in comparison to that of 3 mM Ca2+ which lasted 6 hours. Equimolar concentration of nifedipine did not inhibit the stimulatory effect of BAY K 8644 as compared to the nifedipine only group. The decrease in stimulated CT secretion during long-term exposure to BAY K 8644 is due to desensitization of cells which may be attributed to down-regulation of dihydropyridine receptors. After 12 h exposures to 3 mM Ca2+ alone, BAY K 8644 (10(-6) M) alone or in combination with nifedipine (10(-6) M), CT content decreased below the control level, indicating a decrease in synthesis. Overall cellular protein content was not affected by the test agents. Repetitive exposure of C-cells to BAY K 8644 revealed a desensitization of the stimulatory effect on CT secretion and a decrease in CT cell content.     

Thyrotropin releasing hormone action in pituitary cells. Protein kinase C-mediated effects on the epidermal growth factor receptor

Thyrotropin releasing hormone (TRH) causes phosphatidylinositol bisphosphate hydrolysis to form inositol trisphosphate and diacylglycerol. Since diacylglycerol activates protein kinase C (Ca2+/phospholipid-dependent enzyme), this enzyme may be involved in mediating the physiological response to TRH. Activation of protein kinase C leads to phosphorylation of receptors for epidermal growth factor (EGF) and decreased EGF affinity. The present study examined the effect of TRH on EGF binding to intact GH4C1 rat pituitary tumor cells to test whether TRH activates protein kinase C. Cells were incubated with TRH at 37 degrees C and specific 125I-EGF binding was then measured at 4 degrees C. 125I-EGF binding was decreased by a 10-min treatment with 0.1-100 nM TRH to 30-40% of control in a dose-dependent manner. 125I-EGF binding was not altered if cells were incubated at 4 degrees C, although TRH receptors were saturated or in a variant pituitary cell line without TRH receptors. TRH (10 min at 37 degrees C) decreased EGF receptor affinity but caused little change in receptor density, 125I-EGF internalization, or degradation. When cells were incubated continuously with TRH, there was a recovery of 125I-EGF binding after 24 h. Incubation with the protein kinase C activating phorbol ester TPA caused an immediate (less than 10 min) profound (greater than 85%) decrease in 125I-EGF binding followed by partial recovery at 24 h. Maximally effective doses of TRH and TPA decreased EGF receptor affinity with half-times of 3 min. EGF treatment (5 min) caused an increase in the tyrosine phosphate content of several proteins; prior incubation with TRH resulted in a small decline in the EGF response. GH4C1 cells were incubated with 500 nM TPA for 24 h in order to down-regulate protein kinase C. Protein kinase C depletion was confirmed by immunoblots and the effects of TRH and TPA on 125I-EGF binding were tested. TRH and TPA were both much less effective in cells pretreated with phorbol esters. TRH increased cytoplasmic pH measured with an intracellularly trapped pH sensitive dye after mild acidification with nigericin. This TRH response is presumed to be the result of protein kinase C-mediated activation of the amiloride-sensitive Na+/H+ exchanger and was blunted in protein kinase C-depleted cells. All of these results are consistent with the view that TRH acts rapidly in the intact cell to activate protein kinase C and that a consequence of this activation is EGF receptor phosphorylation and Na+/H+ exchanger activation.     

Dihydropyridine modulators of voltage-sensitive Ca2+ channels specifically regulate prolactin production by GH4C1 pituitary tumor cells

To determine whether hormone synthesis by the GH4C1 pituitary cell line could be regulated by specifically modulating the movement of Ca2+ through voltage-sensitive channels, we have compared the effects of the dihydropyridine Ca2+ channel agonist BAY K8644 and the antagonist nimodipine on hormone production and Ca2+ current in these cells. BAY K8644 elicited, after a 10-15-h lag, a dose-dependent increase in prolactin (PRL) production as determined by measurements of total intracellular and secreted hormone. Over a 72-h period, GH4C1 cells incubated with 300 nM BAY K8644 produced 2-3 times as much total PRL as control cells. The effect on PRL was specific, since BAY K8644 did not increase growth hormone production, cell growth rate, or total cell protein. Exposing GH4C1 cells to BAY K8644 for short periods, up to 90 min, did not induce the delayed increase in PRL production observed with longer incubations. The effects of nimodipine were opposite to those of the Ca2+ channel agonist. PRL production was reduced 85% during 48-h treatment with 200 nM nimodipine, whereas growth hormone production was decreased less than 15%, and cell growth and total protein were unaffected. The actions of these two drugs on PRL production were well correlated with their effects on GH4C1 Ca2+ currents as measured by whole-cell patch-clamp recordings. BAY K8644 enhanced the magnitude of the peak Ca2+ current and shifted the current-voltage relationship such that Ca2+ channels were activated at less depolarized potentials. Nimodipine potently inhibited Ca2+ movement through the non-inactivating channel, while it antagonized the increases elicited by BAY K8644. These results indicate that PRL synthesis by GH4C1 cells can be specifically regulated by agents that enhance or block the movement of Ca2+ through voltage-sensitive channels. They also suggest that hormone synthesis by a secretory cell may be coupled to electrical activity by the opening of Ca2+ channels.     

The role of tyrosine kinase activity in endocytosis, compartmentation, and down-regulation of the epidermal growth factor receptor.

Occupancy-induced down-regulation of cell surface epidermal growth factor (EGF) receptors attenuates signal transduction. To define mechanisms through which down-regulation of this class of growth factor receptors occurs, we have investigated the relative roles of ligand-induced internalization and recycling in this process. Occupied, kinase-active EGF receptors were internalized through a high affinity, saturable endocytic system at rates up to 10-fold faster than empty receptors. In contrast, full length EGF receptors lacking tyrosine kinase activity underwent internalization at a rate independent of occupancy. This "kinase-independent" internalization rate appeared to reflect constitutive receptor internalization since it was similar to the internalization rate of both receptors lacking a cytoplasmic domain and of antibodies bound to empty receptors. EGF internalized by either kinase-active or kinase-inactive receptors was efficiently recycled and was found within endosomes containing recycling transferrin receptors. However, targeting of internalized receptors to lysosomes did not require receptor kinase activity. All receptors that displayed ligand-induced internalization also underwent down-regulation, indicating that the proximal cause of down-regulation is occupancy-induced endocytosis. Tyrosine kinase activity greatly enhances this process by stabilizing receptor association with the endocytic apparatus.     

Analogs of thyrotropin-releasing hormone (TRH): Receptor affinities in brains,spinal cords,and pituitaries of different species

[³H](3-Me-His²) thyrotropin-releasing hormone ([³H]MeTRH) bound to TRH receptors in rodent, rabbit and dog brain and spinal cord (SC), and in rat, sheep, bovine and dog anterior pituitary (PIT) glands, with high affinity (dissociation constants, K_ds=5–9 nM; n=3–4) but to different densities of these sites (B _max range 6–145 fmol/mg protein) (rabbit SC>sheep PITG.pig brain>dog brain>rat brain>bovine and dog PIT). Various TRH analogs competitively inhibited [³H]MeTRH binding in these tissues with a similar rank order of potency: MeTRH>TRH> CG3703RX77368MK-771>TRH Glycinamide>Glu¹-TRHCG3509NVal²-TRH>>>TRH free acid>>>and cyclo-His-Pro, indicating a pharmacological similarity of CNS and pituitary TRH receptors. While most TRH analogs displaced [³H]MeTRH binding with a similar potency in the different species, TRH exhibited a 2-fold lower affinity in the rat and G.pig brain than in other tissues of other species. Similarly, CG3703 was 2.4–4.5 times more active in the rabbit brain than in the rodent and dog brain, and also more potent in the rabbit brain as compared to the sheep PIT. However, MK-771 and RX77368 had a similar affinity for the brain TRH receptors in the different species but RX77368 was 2-fold more active in the SC preparations and 3–4-fold less active in the sheep PIT when compared to the brain homogenates. RX 77368 exhibited the highest affinity for the dog PIT TRH receptor. In contrast, MK-771 showed a similar affinity for the brain, SC and PIT TRH receptor apart from in the rat PIT where it had the highest affinity. Similarly, TRH glycinamide was more active in the dog brain than rodent and rabbit brain. These data suggest that while the rank order of potency of TRH analogs is similar in the species examined, certain analogs appear to be more potent in certain tissues of some species than in others. In addition, the current results have shown that CG3703 is almost equipotent with RX77368 and MK-771 in most species but is substantially more active than its related analog, CG3509 in the brain, SC and PIT. Taken together, these observations may have some relevance to the future clinical applications of these metabolically stabilized TRH analogs.     

The facilitative actions of Bay K 8644 on norepinephrine and KCl-induced contractures of rabbit aortic rings

The effect of the calcium channel agonist BAY K 8644 on the ability of KCl and norepinephrine to induce contractions of rabbit aortic rings has been examined in Krebs-Henseleit buffer containing either 4.0 or 6.8 mM potassium. BAY K 8644 (10(-8) to 10(-6) M) alone induced slowly developing aortic contractures which were 10 (at 4.0 mM potassium) or 20 (at 6.8 mM potassium) percent of the maximum obtainable with norepinephrine. These contractions were not observed in every experiment, but were more likely to occur at 6.8 mM (71% at 10(-6) M BAY K 8644) when compared to 4.0 mM (31% at 10(-6) M BAY K 8644) potassium buffer. BAY K 8644, in either potassium buffer, induced a statistically significant shift to the left in the norepinephrine dose-response curve. The norepinephrine dose-response curve was significantly curvilinear in the presence of 3 X 10(-8) M BAY K 8644 (6.8 mM potassium) and 10(-6) M BAY K 8644 (4.0 mM potassium). Similarly, BAY K 8644 induced sinistral shifts in the KCl dose-response curve with a curvilinear function observed at 3 X 10(-7) M BAY K 8644. These data show that BAY K 8644 is capable of inducing aortic contractures at potassium concentrations significantly lower than previously reported. Furthermore, BAY K 8644 facilitates opening of calcium channels by either potassium or norepinephrine. In contrast to others, our data indicates that BAY K 8644 can affect calcium channels activated by norepinephrine. Finally, our data suggest that the alpha and dihydropyridine receptors are capable of interacting and that occupation of one receptor can affect the action of a compound binding to the other receptor.     

Synergistic stimulation of prolactin release by phorbol ester, A23187 and forskolin

The effects of 12-0-tetradecanoyl-phorbol-13-acetate (TPA), A23187, forskolin and thyrotropin-releasing hormone (TRH) on prolactin release from GH4C1 cells were compared. TPA caused a 2-fold release, maximum after 6 or more min, that was sustained for 30 min or more. A23187 caused only a small and variable response that peaked within 4 to 6 min. Combination of TPA and A23187 caused a rapid 3- to 5-fold increase in release that declined slowly. TRH increased prolactin release 3- to 5-fold, reaching a maximum within 4 min, followed by sustained release at lower rates. Forskolin had little effect by itself, but potentiated release caused either by combined TPA and A23187, or by TRH. These data are consistent with a model in which two branches of the Ca2+ messenger system participate in the action of TRH, a calmodulin branch and a C-kinase branch that interact to cause large amounts of sustained release. Forskolin, by regulating the cyclic AMP content of the cell determines the set point around which the Ca2+ messenger system operates.     

Regulated dimerization of the thyrotropin-releasing hormone receptor affects receptor trafficking but not signaling

To investigate the function of dimerization of the TRH receptor, a controlled dimerization system was developed. A variant FK506 binding protein (FKBP) domain was fused to the receptor C terminus and dimerization induced by incubating cells with dimeric FKBP ligand, AP20187. The TRH receptor-fusion bound hormone and signaled normally. Addition of dimerizer to cells expressing the receptor-FKBP fusion dramatically increased the fraction of receptor running as dimer on SDS-PAGE. AP20187 caused dimerization in a time- and concentration-dependent manner, acting within 1 min. Dimerizer had no effect on TRH receptors lacking the FKBP domain, and its effects were blocked by excess monomeric FKBP ligand. AP20187-induced dimerization did not cause receptor phosphorylation, inositol phosphate production, or ERK1/2 activation, and dimerizer did not alter signaling by TRH. Induced dimerization did, however, alter TRH receptor trafficking. TRH promoted greater receptor internalization in cells treated with AP20187 but not monomeric ligand, based on loss of surface binding sites and immunostaining. Dimerization increased the rate of internalization of TRH receptors and decreased the apparent rate of receptor recycling. AP20187 enhanced the small amount of TRH-induced receptor internalization when the receptor-FKBP fusion protein was expressed in cells lacking beta-arrestins. The results show that controlled dimerization of the TRH receptor potentiates hormone-induced receptor trafficking.     

Chlordiazepoxide is a competitive thyrotropin-releasing hormone receptor antagonist in GH3 pituitary tumour cells

Addition of thyrotropin-releasing hormone (TRH) to [3H]-inositol pre-labelled GH3 pituitary tumour cells suspended in medium containing 10mM lithium chloride led to a rapid diminution in cellular [3H]-inositol and increase in [3H]-inositol 1-phosphate (InslP), [3H]-inositol bisphosphate (InsP2) and [3H]-inositol trisphosphate (InsP3). In the presence of the benzodiazepine tranquillizer, chlordiazepoxide, the TRH concentration-response curves for these effects were shifted to the right in a parallel fashion. The Ki for chlordiazepoxide in inhibiting all four responses was 1.5 X 10(-5)M. Chlordiazepoxide did not inhibit the small bombesin-induced rise in [3H]-InslP. Another benzodiazepine, diazepam, was less active. The TRH-induced rise in cytosolic free calcium monitored in Quin-2-loaded GH3 cells was also blocked by chlordiazepoxide in a competitive manner, while that induced by high K+-induced depolarisation was unaffected. It is suggested that chlordiazepoxide acts as a competitive antagonist at the level of the TRH receptor.