Involvement of alpha 1-adrenergic receptors in the inhibitory effect of catecholamines on the thyrotropin-induced release of thyroxine by the mouse thyroid.

TSH enhanced the release of T4 from the mouse thyroid incubated in vitro. Norepinephrine, a nonspecific alpha-adrenergic agonist, and methoxamine, an alpha 1-agonist, inhibited the TSH-stimulated release of T4 at 10(-4) and 10(-5) M, whereas clonidine, an alpha 2-agonist, exerted a weak inhibition. The inhibitory effect of 10(-5) M norepinephrine on the T4 release stimulated by TSH was prevented by prazosin, an alpha 1-antagonist, at concentrations higher than 10(-7) M, whereas yohimbine, an alpha 2-antagonist, exerted weak activity in antagonizing the inhibition induced by norepinephrine. Norepinephrine, methoxamine, and clonidine did not significantly reduce the cAMP accumulation stimulated by TSH in the mouse thyroid incubated in vitro. These findings in the mouse thyroid indicate that catecholamines act by way of alpha 1-adrenergic receptors to suppress TSH-stimulated release of T4 without reducing the cAMP levels stimulated by TSH in the mouse thyroid.

Afferent fibers mediate the increase of met-enkephalin elicited in rat spinal cord by localized pain.

Met-enkephalin levels were measured in various spinal cord regions of rats chronically suffering from the inflammation of a single paw following a treatment with Freund's adjuvant. The results indicate that chronic localized pain induces a selective increase of met-enkephalin immunoreactive material (ME-IR) in the dorsal horn of the spinal cord segment which receives a direct projection from the inflamed paw. In order to gain information on the functional meaning of these data, either the plexus brachialis or the sciatic nerve were sectioned peripherally before inducing inflammation. Denervation prevented the increase of ME-IR concentration induced by the injection of Freund's adjuvant. Our observations suggest that chronic localized pain in a limb induces a change in ME-IR content which is selective for the spinal cord segment receiving a direct projection from the inflamed paw. This increase depends on an intact innervation.

Involvement of alpha 1-adrenergic receptors in stimulation of phosphatidylinositol metabolism by catecholamines in mouse thyroids.

The effects of alpha-adrenergic agonists and thyroid stimulating hormone on the incorporation of radioactive phosphate into phosphatidylinositol were investigated in mouse thyroids in vitro. The incorporation of 32P orthophosphate into phosphatidylinositol was stimulated by thyroid stimulating hormone, norepinephrine (a mixed alpha 1- and alpha 2-adrenergic agonist), methoxamine and phenylephrine (alpha 1-agonists) and slightly by clonidine and oxymetazoline (alpha 2-agonists) but not by isoproterenol (beta-agonist). Prazosin (alpha 1-antagonist) inhibited the stimulation by norepinephrine of 32P incorporation into phosphatidylinositol, but yohimbine (alpha 2-antagonist) was less effective. Although norepinephrine inhibits the thyroid stimulating hormone-induced release by activating alpha-, especially alpha 1-adrenoceptors in mouse thyroids [M. L. Maayan et al., Metabolism 26, 473 (1977); M. L. Maayan et al., Endocrinology 101, 284 (1977); T. Muraki et al., Endocrinology 110, 51 (1982)] alpha 1-agonists did not decrease the stimulation of turnover elicited by thyroid stimulating hormone and did not have additive action with it. These results suggest that (1) the stimulation of phosphatidylinositol turnover of mouse thyroids elicited by adrenergic agonists is mediated by activation of alpha 1-adrenoceptors and (2) the inhibitory effect of norepinephrine on the thyroid stimulating hormone-induced release of thyroxine is not mediated by norepinephrine-inhibition of phosphatidylinositol-turnover stimulated by thyroid stimulating hormone.

Potentiation of prostaglandin E1-stimulated cAMP formation by 12-O-tetradecanoylphorbol-13-acetate in BALB/c mouse 3T3 cells.

Prostaglandin E1 (PGE1: 0.1-100 microM), forskolin (0.1-100 microM), and cholera toxin (20 ng/ml) stimulated cAMP formation of BALB/c 3T3 cells. The pretreatment of the cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced PGE1 (10 microM)-stimulated cAMP formation in a concentration- and a time-dependent manner. If the cells were pretreated with TPA (0.1 microM) for only 1 hr, the above augmentation was not observed. Maximal enhancement was observed by pretreatment of the cells for 5 hr with 0.1 microM TPA. Basal cAMP formation was not affected by TPA pretreatment. Other tumor promoters, such as teleocidin and mezerein, showed a potentiating effect similar to that of TPA on the PGE1-stimulated cAMP formation. However, phorbol which is not a tumor promoter, failed to potentiate PGE1 action significantly. These results suggest that the above TPA action may share some common mechanisms with the tumor-promoting action of this agent. On the other hand, the forskolin- and cholera toxin-stimulated cAMP formations were not changed by pretreatment of the cells with TPA. Therefore, our results indicate that the potentiating action of TPA on PGE1-stimulated cAMP formation in 3T3 cells is not due to the activation of the catalytic unit or the stimulatory guanine nucleotide binding protein (Ns) of adenylate cyclase (AC) system by this agent. It is highly likely that TPA induces some alterations on PGE1 receptors or on PGE1 receptor-Ns coupling systems and consequently induces an augmentation of PGE1-stimulated cellular cAMP response.

Strain difference in morphine-induced increase in plasma cyclic AMP and cyclic GMP levels in relation to locomotor activity in male mice.

The effects of morphine on plasma cyclic nucleotide levels and on locomotor activity were investigated in four inbred strains of male mice. Morphine increased both cyclic AMP and cyclic GMP levels as well as locomotor activity in C57BL/6N mice. In BALB/cAnN mice, morphine increased plasma cyclic GMP levels and motor activity without changes in plasma cyclic AMP levels. In C3H/HeN mice, morphine increased plasma cyclic GMP levels without changing cyclic AMP levels and motor activity, but neither plasma cyclic nucleotide levels nor motor activity were increased by morphine in DBA/2N mice. Epinephrine and carbachol increased plasma cyclic AMP and cyclic GMP levels, respectively, in both C57BL and DBA mice. These results show that there is a significant strain difference in the effects of morphine on plasma cyclic nucleotide levels as well as motor activity. The major cause of strain difference in the effects of morphine on cyclic nucleotide levels is unlikely to be due to the difference in the regulation of adenylate cyclase linked to adrenoceptors or that of guanylate cyclase connected with cholinoceptors.

Chronic ethanol changes opiate receptor function in rat striatum.

Ethanol produces supersensitivity of striatal delta-opiate receptor sites labeled by [3H]DADLE and [3H]etorphine. The impairment may be ascribed to the diminished enkephalin release detected in rat striatum after chronic ethanol consumption. On the contrary, a lower affinity of striatal mu-opiate receptors results after same ethanol exposure. In fact, the Kd values of [3H]Met-enkephalin and [3H]DHM are enhanced when measured in striata of ethanol-dependent rats. The diverse sensitivity of the various classes of opiate receptors to ethanol may be ascribed to different ethanol effects on enkephalinergic transmission.

Effects of GTP and sodium on rat striatal dopamine receptors labeled with lisuride.

[3H]Lisuride binding to rat striatal membranes appeared to be stereospecifically displaced by the dopamine antagonist butaclamol. Sodium increased the number of [3H]lisuride binding sites (Bmax) without changing the dissociation constant (Kd). GTP did not affect [3H]lisuride binding characteristics, either with or without sodium. These results suggest that dopamine receptor sites labeled by lisuride are at least in part sodium-dependent, possibly the D2-receptors not involved in adenylate cyclase stimulation.

Improvement of anemia in W/WV mice by recombinant human erythropoietin (rHuEPO) mediated through EPO receptors with lowered affinity.

We studied the effects of recombinant human erythropoietin (rHuEPO) on anemic W/WV mice which manifested severe anemia accompanied by mutation of the W gene encoding tyrosine kinase type receptor (c-kit gene) of bone marrow hematopoietic cells. Nine-week-old male W/WV mice or normal littermates (+/+) were used. Since serum EPO concentration in W/WV mice increased in proportion to severity of anemia, EPO production in the kidneys of these animals was found to be regulated normally. Hematocrit in +/+ mice increased and a maximal response was also obtained with 2,000 IU/kg of rHuEPO. On the other hand, hematocrit in W/WV mice increased in a dose-responsive manner by administration with 2,000 and 10,000 IU/kg, showing different responses to rHuEPO in these two types of mice. The responsiveness of W/WV mice to rHuEPO was low in terms of increases in erythroblastic precursor cells (CFU-E), and immature cells in the bone marrow. Scatchard analysis of the specific binding of 125I-rHuEPO against bone marrow cells revealed that the different responsiveness to rHuEPO between W/WV and +/+ mice may be correlated with differences in affinity of EPO receptor of bone marrow cells in these mice. From these results, a high dose of rHuEPO is capable of improving the anemia in W/WV mice that had EPO receptors with lowered affinity, indicating the possible effectiveness of rHuEPO in anemic patients with EPO receptor abnormality.

Effect of morphine on the tissue cyclic AMP and cyclic GMP content in two strains of mice.

The effect of morphine on the cyclic (c) AMP and cyclic (c) GMP concentrations in several organs, and its reversal by naloxone have been investigated in C57BL and DBA strains of mice. Morphine increased the cAMP contents in lungs and muscle, and the cGMP contents in lungs, intestine, heart, liver and muscle in a naloxone-reversible way in C57BL mice only. This is consistent with our previous observation that morphine increased plasma cyclic nucleotide levels in C57BL mice, whereas such an increase was marginal in the DBA strain. These results show that there is a strain difference in the effect of morphine on tissue cyclic nucleotide contents and the possible origin of the plasma cyclic nucleotides which are increased by morphine.

Effect of a chymotrypsin-like inhibitor, TPCK, on histamine release from cultured human mast cells.

The involvement of endogenous proteases in the secretory process from human mast cells remains to be clarified. A chymotrypsin-like protease inhibitor, N-tosyl-L-phenylalanylchloromethyl ketone (TPCK), blocked both FceRI- and A23187-mediated histamine release from cultured human mast cells at concentrations above 1 microM. At 10 microM, the concentration that completely inhibited FceRI-mediated histamine release, TPCK did not inhibit the chymase activity of the lysate or that in intact cells. The addition of TPCK to cells 30 min before challenge did not affect FceRI- or A23187-mediated Ca2+ mobilization. These findings suggest that a TPCK-sensitive molecule distinct from chymase is involved in a late stage of the process of histamine release from mast cells in man.

Effects of lipoxygenase and cyclooxygenase inhibitors on the induction of ornithine decarboxylase by 12-O-tetradecanoylphorbol-13-acetate and isoproterenol in mouse tissues in vivo.

I.p. administration of 12-O-tetradecanoylphorbol-13-acetate (TPA) (400 micrograms/kg) caused a remarkable increase in ornithine decarboxylase (ODC) activity in CD-1 mouse liver (8.3-fold), spleen (17.8-fold), kidney (4-fold), lung (7.7-fold) and brain (2.7-fold). TPA induced an increase in ODC activity in liver, spleen and kidney in a dose-dependent manner (100-800 micrograms/kg). The putrescine contents of these tissues were also increased by TPA injection. BW755C, an inhibitor of cyclooxygenase and lipoxygenase, prevented the TPA-induced increase in ODC activity in liver, spleen and kidney in a dose-dependent manner. AA861-a selective lipoxygenase inhibitor, also showed the inhibition of TPA-induced increase in ODC activity in these tissues. Significant inhibition was observed either by BW755C or AA861 at the dose of 30 mg/kg. On the other hand, indomethacin, a selective cyclooxygenase inhibitor, enhanced the TPA-induced increase in ODC activity in these tissues dose-dependently. Significantly enhancement was observed at 3 mg/kg for liver and spleen and 1 mg/kg for kidney. The subcutaneous administration of isoproterenol (1 mg/kg) caused an increase in ODC activity in both liver (11-fold) and spleen (3.4-fold). Both AA861 and BW755C failed to inhibit the isoproterenol-induced increase in ODC activity in these tissues. These results indicate that product(s) of lipoxygenase pathway play an important role in ODC induction caused by TPA in liver, spleen and kidney, while the lipoxygenase pathway does not play an essential role in the isoproterenol-induced increase in ODC activity.

Lipoxygenase inhibitors and cyclic AMP-mediated insulin secretion caused by forskolin, theophylline and dibutyryl cyclic AMP.

Forskolin caused a marked and a concentration-dependent elevation of cyclic AMP content in isolated pancreatic islets (EC50, 10 microM). Cyclic AMP level reached a plateau within 30 min after the addition of 10 microM forskolin. In a low glucose (3.3 mM) medium, forskolin induced slight but significant insulin secretion in a concentration-dependent manner (EC50, 0.3 microM). When the glucose concentration was increased to 5.5 mM, marked enhancement of insulin secretion was observed with forskolin (EC50, 0.5 microM). Lipoxygenase inhibitors, such as nordihydroguaiaretic acid, 3-amino-1-(trifluoromethylphenyl)-2-pyrazoline and 1-phenyl-3-pyrazolidinone failed to affect the forskolin-stimulated cyclic AMP generation. The selective cyclooxygenase inhibitor indomethacin also had no influence on forskolin-stimulated cyclic AMP generation. Insulinotropic effects of forskolin, however, were suppressed by these lipoxygenase inhibitors but not by indomethacin. Both nordihydroguaiaretic acid and 1-phenyl-3-pyrazolidinone also prevented the insulinotropic effects of theophylline and dibutyryl cyclic AMP, whereas indomethacin failed to inhibit them. It seems conceivable that a lipoxygenase product(s) is involved in the insulin secretory process distal to cyclic AMP generation, or that alternatively a lipoxygenase product(s) is permissively involved in the insulin secretory process independently from the cyclic AMP-mediated process.

Effects of KRN4884 (a novel K+ channel opener), levcromakalim, nilvadipine and propranolol on endothelin-1-induced heart disorders in anesthetized rats.

The effects of KRN4884 (5-amino-N-[2-(2-chrolophenyl)ethyl]-N'-cyano-3-pyridinecarboxa midine), a novel K+ channel opener, on the electrocardiogram changes caused by the intracoronary administration of endothelin-1 (ET-1) were studied in anesthetized rats and compared with the effects of levcromakalim, a K+ channel opener; nilvadipine, a Ca2+ antagonist; and propranolol, a beta-adrenoceptor antagonist. KRN4884 (50 microg/kg, i.v.) and levcromakalim (300 microg/kg, i.v.) inhibited the ST segment elevation and the development of arrhythmias induced by ET-1 (5 microg, i.c.) and decreased the incidence of death. Nilvadipine (300 microg/kg, i.v.) and propranolol (1000 and 3000 microg/kg, i.v.) each prevented the ST segment elevation, but the suppressions of the occurrence of arrhythmias produced by nilvadipine and propranolol were less than that shown by KRN4884. KRN4884 (30 and 50 microg/kg, i.v.), levcromakalim (100 and 300 microg/kg, i.v.) and nilvadipine (100 and 300 microg/kg, i.v.) significantly decreased the mean blood pressure in a dose-dependent manner, but propranolol did not. The heart rate was decreased by nilvadipine (100 and 300 microg/kg, i.v.) and propranolol (1000 and 3000 microg/kg, i.v.), but was not affected by KRN4884 (30 and 50 microg/kg, i.v.) or levcromakalim (100 and 300 microg/kg, i.v.). These results suggest that pretreatments with KRN4884 and levcromakalim are more effective on ET-1-induced electrocardiogram changes than those with nilvadipine and propranolol.

Characterization of receptor for granulocyte colony-stimulating factor on human circulating neutrophils.

We have made a mutein of human G-CSF with more stable, and potent biological activity. Using 125 I-labeled mutein human G-CSF, high affinity binding sites were identified on human circulating neutrophils. Receptor number per cell was 560 with a Kd of 250 pM. The human G-CSF receptor was identified as a single subunit protein of Mr approximately 150,000.

Granulocyte colony-stimulating factor (G-CSF) receptors on acute myeloblastic leukaemia cells and their relationship with the proliferative response to G-CSF in clonogenic assay.

The number and the affinity of granulocyte colony-stimulating factor (G-CSF) receptors expressed by blast cells in acute myeloblastic leukaemia (AML) were determined using radiolabelled recombinant human G-CSF (rhG-CSF). Eighteen of 20 patients demonstrated specific binding, and Scatchard analysis revealed a single class of high affinity (Kd 15-130 pM) G-CSF receptors on the AML blasts. The number of G-CSF receptors varied from 55 to 1,200 per cell (mean 278). In the remaining two patients, specific binding was not observed. The number of G-CSF receptors did not differ significantly between various AML subtypes, but the mean receptor number was the highest on type M2 blasts. A chemical cross-linking study revealed that the G-CSF receptor has an approximate molecular weight of 140,000. Autoradiography showed heterogeneity of the distribution of G-CSF receptors on the AML blasts obtained from a single patient. The number of colonies stimulated by the addition of rhG-CSF varied from 0 to 566 per dish, and blast colony formation was observed in eight of 20 patients. The population mean of G-CSF receptor number expressed by blasts that formed colonies on stimulation with rhG-CSF was significantly higher than that on blasts which did not form colonies. These results suggest that a proliferative response of AML blasts to G-CSF may be predicted when the blasts express a large number of G-CSF receptors. Accordingly, it may be safer to restrict the clinical use of G-CSF to AML patients who have blasts with a low G-CSF receptor expression and no response to G-CSF in blast colony assay.

Mutant protein of recombinant human granulocyte colony-stimulating factor for receptor binding assay.

A new mutant protein of recombinant human granulocyte colony-stimulating factor (rhG-CSF) was produced for the studies on receptors for human G-CSF. The mutant protein [(Tyr1, Tyr3]rhG-CSF), the biological activity of which was almost equal to that of rhG-CSF, was prepared by the replacement of threonine-1 and leucine-3 of rhG-CSF with tyrosine. The radioiodinated preparation of the mutant protein showed high specific radioactivity and retained full biological activity for at least 3 weeks. The binding capacity of the radioiodinated ligand was compared with that of [35S]rhG-CSF. Both radiolabeled ligands showed specific binding to murine bone marrow cells. Unlabeled rhG-CSF and human G-CSF purified from the culture supernatant of the human bladder carcinoma cell line 5637 equally competed for the binding of labeled rhG-CSFs in a dose-dependent manner, demonstrating that the sugar moiety of human G-CSF made no contribution to the binding of human G-CSF to target cells. In contrast, all other colony-stimulating factors and lymphokines examined did not affect the binding. Scatchard analysis of the specific binding of both labeled ligands revealed a single class of binding site with an apparent dissociation constant (Kd) of 20-30 pM and 100-200 maximal binding sites per cell. These data indicate that the radioiodinated preparation of the mutant protein binds the same specific receptor with the same affinity as [35S]rhG-CSF. The labeled mutant protein also showed specific binding to human circulating neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of cord-blood-derived human mast cells cultured in the presence of Steel factor and interleukin-6.

We generated > 10(7) mast cells by culturing 10(7) cord blood mononuclear cells for > 10 weeks in the presence of Steel factor, interleukin-6 and prostaglandin E2. 99% of the cultured cells had tryptase-positive granules, while 18% had chymase-positive granules. Cultured mast cells contained 3.6 micrograms histamine and 3.5 micrograms tryptase per 10(6) cells. Cells sensitized with 1 microgram/ml human IgE released 58.5% histamine and 1.55 ng tumor necrosis factor (TNF)-alpha per 10(6) cells when challenged with 1 microgram/ml antihuman IgE, whereas the control cells spontaneously released 3.7% histamine and 0.18 ng TNF-alpha. Analysis for surface antigens revealed that cultured mast cells expressed the following CD molecules: 9, 13, 14, 29, 33, 38, 43, 44, 45RA, 45RB, 46, 47, 48, 49d, 50, 51, 53, 54, 55, 58, 59, 60, 61 and 117 (c-Kit). Taken together, these cultured cells seem to be functionally mature mast cells.

Interferon-gamma promotes the survival and Fc epsilon RI-mediated histamine release in cultured human mast cells.

We examined the effects of interferon-gamma (IFN-gamma) on 100% pure human mast cells generated in suspension cultures of umbilical cord blood mononuclear cells in the presence of stem cell factor (SCF) and interleukin-6 (IL-6). When mast cells were suspended in serum-free medium without any cytokine after the withdrawal of SCF and IL-6, they died over a period of 5 days because of apoptosis. IFN-gamma in the cultures suppressed apoptosis and prolonged their survival in a dose-dependent manner. This survival-promoting effect of IFN-gamma was blocked by neutralizing antibodies to IFN-gamma or to IFN-gamma receptor (IFN-gamma R). When mast cells were incubated with IFN-gamma in serum-free medium for more than 4 hr during sensitization, immunoglobulin E (IgE)/anti-IgE antibody-induced histamine release was effectively enhanced. Polymerase chain reaction (PCR) amplification of the alpha-chain of IFN-gamma R (IFN-gamma R alpha) yielded products of the correct size predicted from the sequence of the receptor. In addition, flow cytometry using anti-IFN-gamma R monoclonal antibodies (mAbs) indicated that these mast cells bear IFN-gamma R on their surface. These findings suggested that IFN-gamma activates human mast cells via specific receptors in certain aspects of inflammatory reactions.

Identification and characterization of receptors for granulocyte colony-stimulating factor on human placenta and trophoblastic cells.

Since radioiodination of human granulocyte colony-stimulating factor (G-CSF) is difficult, we synthesized a mutein of human G-CSF that retains full biological activity and receptor-binding capacity for at least 2 weeks after radioiodination. Receptors for human G-CSF were characterized in the plasma membrane fraction from the human term placenta (human placental membranes) and trophoblastic cells by using the 125I-labeled mutein of human G-CSF (KW-2228). The specific binding of 125I-labeled KW-2228 to placental membranes was pH-dependent, with maximal specific binding at pH 7.8; it increased linearly with protein to 3.7 mg of protein per ml and was both time- and temperature-dependent, with maximal binding at 4 degrees C after a 24-hr incubation. When we examined the ability of hematopoietic growth factors to inhibit 125I-labeled KW-2228 binding, we found that KW-2228 and intact human G-CSF inhibited 125I-labeled KW-2228 binding, whereas erythropoietin or granulocyte-macrophage colony-stimulating factor did not. Scatchard analysis revealed a single receptor type with a Bmax of 210 fmol/mg of protein and a Kd of 480 pM. The human G-CSF receptors on human placental membranes were shown to consist of two molecular species of 150 kDa and 120 kDa that could be specifically cross-linked to 125I-labeled KW-2228. Human trophoblastic cells, T3M-3, also possessed a single receptor for G-CSF with a Bmax of 533 receptors per cell and a Kd of 390 pM. Thus we have identified the receptor for human G-CSF on human placental membranes and trophoblastic cells, and the presence of this receptor in these membranes suggests that human G-CSF plays some role in the feto-placental unit during human development.

The role of transforming growth factor-beta in PEG-rHuMGDF-induced reversible myelofibrosis in rats.

Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) injected at a suprapharmacologic dose (100 microg/kg) daily for 5 d in normal rats caused marked increases in marrow megakaryocytes and platelet counts at 6-8 d followed by gradual decreases to control levels at 10-20 d. Interestingly, in addition to the expected thrombopoiesis, PEG-rHuMGDF was associated with myelofibrosis with a predominance of reticulin fibres at day 10 followed by complete normalization by day 20. At 6-8 d, the levels of transforming growth factor-beta1 (TGF-beta1) in the extracellular fluid of the marrow, the platelet poor plasma, and the platelet extract were increased 23-, 7- and 2-fold, respectively. The elevated levels of TGF-beta1 were gradually reduced to baseline levels at 13-20 d in accordance with the normalization of myelofibrosis and thrombopoiesis. An ultrastructural analysis showed that large fragments of megakaryocytes were deposited in the marrow parenchyma of PEG-rHuMGDF-treated rats at day 6. PEG-rHuMGDF administration at pharmacologic doses (1 and 10 microg/kg) did not induce the deposition of reticulin fibres in the marrow. These findings suggest that TGF-beta1 leaked from megakaryocytes is involved in the development of the PEG-rHuMGDF-induced myelofibrosis and that this is a reversible process related to the regulation of the excess production of platelets.