Molecular cloning of quail thyroid-stimulating hormone (TSH) beta subunit

Thyroid-stimulating hormone (TSH) beta subunit cDNA was obtained from Japanese quail (Coturnix japonica) by PCR and its nucleotide sequence was determined. The cDNA encodes a putative signal peptide and a mature protein consisting of 20 and 114 amino acids, respectively. The amino acid sequence of quail TSHbeta subunit shows homologies of 67-69% in mammalian species, 58% in amphibian and 43-49% in teleost fish. Comparison of the amino acid sequence with TSHbeta subunits of other species reveals some differences in several regions responsible for its biological functions and characteristic features of the avian TSHbeta subunit, suggesting that the functional domains have diverged cooperatively between the hormone and its receptor during evolution.     

The human thyrotropin (TSH) receptor in a TSH binding inhibition assay for TSH receptor autoantibodies

Seven years after the molecular cloning of the human TSH receptor (TSHR), the porcine TSHR remains in general use in the TSH binding inhibition (TBI) assay for autoantibodies to the TSHR. We compared porcine and recombinant human TSHR in two types of TBI assays: one using intact Chinese hamster ovary cells expressing the recombinant human TSHR on their surface, and the other using soluble receptors extracted from these cells with detergent. In the intact cell TBI assay, monolayers expressing large numbers of TSHR were less effective than cells expressing few receptors. These findings are consistent with the very low concentration of TSHR autoantibodies in serum. Binding of [125I]human TSH was about 5-fold lower than that of [125I]bovine TSH to the intact cells. Nevertheless, TBI values with the two ligands were similar for most sera. However, a few sera produced greater inhibition of human than of bovine TSH binding. In the solubilized human TSHR TBI assay, in contrast to the intact cell TBI assay, cells expressing very large number of TSHR were an excellent source for detergent extraction of soluble human TSHR, but only if the cells were extracted while still on the dish and not after scraping. A 10-cm diameter dish of cells provided TSHR for 100-200 replicate determinations when substituted for solubilized porcine TSHR in a commercial TBI kit. TBI values in serum from 30 individuals with suspected Graves' disease correlated closely when tested with solubilized human and porcine TSHR (r = 0.954; P < 0.001). However, 2 sera that were negative with the porcine TSHR were positive with the human TSHR. TBI and thyroid-stimulating activity in these sera correlated weakly regardless of whether the TBI used human or porcine TSHR. These findings open the way to a practical TBI assay using recombinant human TSHR.     

Pharmacological characterization of tachykinin-induced intracellular calcium rise in a human NK2 receptor transfected cell line

We have examined the effect of various natural and synthetic tachykinins on the steady state Ca(++)-rise ([Ca++]i) in transfected chinese hamster ovary cells expressing recombinant human Neurokinin 2 (NK2) receptors. The rank order of potency with natural tachykinins was NeurokininA > Neurokinin B > Eledoisin > Physaelamin > substance P. The selective NK2 agonist, [beta-Ala8]NKA(4-10) was very potent, with an EC50 value of 4.83 x 10(-9) M whereas Senktide, MePhe7NKB and Sar9, (MetO2)11 substance P, selective NK3 and NK1 agonists, respectively, did not have any effect on [Ca++]i in hrNK2CHO cells, suggesting a selective and preferential recognition and activation of NK2 receptors in these cells. (+/-) SR 48968, a selective NK2 antagonist, abolished the beta-AlaNKA-induced [Ca++]i with an IC50 value of 0.7 nM. Two other peptidic NK2 antagonists, MEN 10376 and L-658977, were less active with IC50 values of 49 nM and 5.29 microM, respectively. In contrast, (+/-) CP-96,345 and (+/-)CP-99,994 and RP 67580, all selective NK1 antagonists, did not have any effect on the beta-AlaNKA-induced [Ca++]i in hrNK2CHO cells (+/-) SR 140333, a potent and selective NK1 antagonist, had a 35% inhibition under similar conditions. These data demonstrate a high selectivity and sensitivity to NK2 receptor mediated [Ca++]i in rhNK2R-CHO cells and may be of value as a rapid, selective test of drug action at the human NK2 receptors in vitro.     

Growth hormone regulation of lipid metabolism in cells transfected with growth hormone receptor cDNA

We have studied, by histological methods, cytological progression, frequency and distribution of apoptosis in the external granular layer of the cerebellum after whole-body irradiation of 14-day-old rats by gamma-rays from 60 Co. After acute exposure to 0.25, 0.5, 1.5 and 3 Gy (18 cGy/min), the duration of the apoptotic process gradually increased with dose from 6-9 h after 0.25 Gy, to > 24 h after 3 Gy. Up to 1 Gy, maximal frequency was found 6 h after exposures, and at this postirradiation time a linear increase in apoptosis with dose was observed. No effect of dose-rate on apoptosis induction could be demonstrated 6 h after delivering 1 Gy at dose-rates from 2.2 to 18 cGy/min. Continuous irradiation at 1.8 cGy/h induced a gradual increase of apoptosis that remained at a plateau value of about 3% from 15 to 29 h (controls 0.12%, SD = 0.07) and then gradually decreased to 1% at 53 h. At this time the mitotic index was similar to that measured in controls. Apoptosis occurring 3 h after acute irradiation, confined to proliferative cells, was only observed for doses of 1.5 and 3 Gy.     

Expression of the human TSH receptor in a human thyroid carcinoma cell line that lacks an endogenous TSH receptor: growth inhibition by cAMP

The sexually dimorphic pattern of GH secretion regulates the expression of several steroidogenic enzymes in rat liver, including a male-specific 3 beta-hydroxysteroid dehydrogenase/delta 5-->4-isomerase (3 beta HSD). Recently, we identified male-specific isoforms of immunoreactive 3 beta HSD in mouse liver [42 kilodaltons (kDa)] and gonads (47 kDa). To test whether GH can regulate the expression of these murine 3 beta HSDs, endogenous forms of 3 beta HSD were studied in transgenic mice expressing heterologous GH transgene products. Mice from five transgenic lines were used; two expressed GH transgenes encoding the phosphoenolpyruvate carboxykinase (PEPCK) promoter fused to either the human (h) GH (somatogenic and lactogenic) or bovine (b) GH (somatogenic) structural genes, and three expressed GH transgenes encoding the mouse metallothionein-1 (MT1) promoter fused to the hGH, hGH variant (hGHv), or bGH structural genes. Control mice were normal nontransgenic littermates. Expression of a male-specific (42 kDa) isoform of hepatic 3 beta HSD is dramatically suppressed in all transgenic mouse lines, as detected on Western immunoblots, without affecting a 47-kDa isoform expressed in livers of both male and female mice. This negative regulation was not observed in mouse kidney, which normally expresses two 3 beta HSD isoforms (in both sexes) with molecular masses similar to those in liver. Considering that PEPCK and MT1 promoters direct expression of GH fusion genes in both tissues, the inhibition of hepatic, but not renal, 3 beta HSD immunoreactivity suggests that GH affects sex-specific, rather than tissue-specific, expression of 3 beta HSD. As in the liver, sex-specific expression of 3 beta HSD in the testis is also suppressed by heterologous GH, but with one notable difference. Only human-derived GH (MT1-hGH and MT1-hGHv) effectively inhibits expression of the 47-kDa sex-specific isoform of testicular 3 beta HSD, without affecting the 44-kDa isoform expressed in gonads of both male and female mice. These results suggest that the negative effects of heterologous GH on sex-specific 3 beta HSDs may be mediated by PRL receptors in the testis and GH receptors in the liver. PEPCK-GH transgenes had little effect on testicular 3 beta HSD, possibly because this promoter (unlike MT1) is relatively inactive in this tissue. In the liver of male transgenics (PEPCK-hGH), loss of the sex-specific (42-kDa) 3 beta HSD has little effect on the Km for dehydroepiandrosterone (DHEA; 11 microM) compared with that in normal controls (16 microM).(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of brief illumination on intracellular free calcium concentration in cells with 5-aminolevulinic acid-induced protoporphyrin IX synthesis

The effect of illumination on intracellular free calcium concentration, [Ca2+]i, was studied in a cell line (WiDr cells) derived from a primary adenocarcinoma of the rectosigmoid colon. In these cells the biosynthesis of protoporphyrin IX was stimulated by 5-aminolevulinic acid to reach levels of 600-700 pmol of protoporphyrin IX per mg cell protein. A brief (1-min) exposure of the cells to light (70% of light energy at 340-380 nm) resulted in an increase in [Ca2+]i. This increase was not reversible over a period of at least 20 min following illumination. Elevation of [Ca2+]i most probably represented an influx of calcium ions from the medium to the cell, since it was completely abolished in the presence of extracellular EGTA. The increased [Ca2+]i did not reflect general membrane damage, as determined by trypan blue staining as well as measurement of the intercalation of ethidium bromide into cellular DNA, and neither did the sustained elevation of [Ca2+]i lead to any substantial loss of clonogenicity following illumination of protoporphyrin-containing cells. Together these results indicate that an increased [Ca2+]i level is not per se a cause of cell death during photodynamic therapy.     

Two novel mutations in the thyrotropin (TSH) receptor gene in a child with resistance to TSH

The TSH receptor is a G protein-coupled receptor that mediates the effects of TSH in thyroid development, growth, and synthetic function. We report here that a child with features of TSH resistance, including markedly increased serum TSH concentrations and low normal thyroid hormone levels, is a compound heterozygote for two novel mutations in the TSH receptor gene. One allele has a G to A transition corresponding to an arginine to glutamine change at codon 109 (R109Q) in the extracellular domain of the receptor. The other allele has a G to A transition corresponding to a premature termination codon at tryptophan 546 (W546X) in the fourth transmembrane segment. Each parent is heterozygous for one mutation, and both parents have normal thyroid function. Cells transiently transfected with the R109Q mutant exhibited reduced membrane binding of [125I]TSH and impaired signal transduction in response to TSH. In contrast, the W546X mutant was nonfunctional, with negligible membrane radioligand binding. Our findings indicate that a single normal TSH receptor allele is sufficient for normal thyroid function, but that the compound abnormality in the proband leads to TSH resistance.     

Effect of pteridine derivatives on intracellular calcium concentration in human monocytic cells

Pteridines are heterocyclic compounds which are synthesized and released by human monocytes/macrophages following stimulation by interferon-gamma. Their concentration in various body fluids proved to be indicative for the stimulation of the cellular immune system, and determination of pteridines has become an important diagnostic tool. We show that pteridine derivatives, namely neopterin (N), 7,8-dihydroneopterin (NH2), and 5,6,7,8-tetrahydrobiopterin (BH4) increase intracellular calcium (Cai) in human monocytic cells. Significant increases of Cai are observed at 10 nmol/l NH2, at 100 nmol/l BH4 and at 1 mol/l N, i.e. at concentrations encountered in vivo. At a concentration of 1 mumol/l, Cai is increased (from a control value of 145 +/- 7 nmol/l) to 464 +/- 62 nmol/l (NH2), 340 +/- 41 nmol/l (BH4) and 344 +/- 46 nmol/l (N), respectively. The increase of Cai depends on the presence of extracellular calcium and is likely to be due to activation of a calcium channel. We show that the absence of extracellular calcium or the addition of lanthanum ions to the extracellular fluid fully reverses the pteridine-induced increase of Cai. According to these observations, pteridines may mimic the effects of other inflammatory mediators on monocytic cells and seem to be involved in the crosstalk of immunocompetent cells.     

Relationship between intracellular free calcium concentrations and the intracellular development of Toxoplasma gondii

We measured intracellular free calcium concentrations ([Ca++]i) in the subcellular compartments of Toxoplasma gondii infected living cells using microspectrofluorometry and Indo-1 staining. [Ca++]i mapping was defined in infected and uninfected cells and in the neoformed parasitophorous vacuole (PV) 24 and 48 hr after parasite inoculation. At 24 hr after infection, a [Ca++]i gradient (PV/cytoplasm) was observed in favor of the PV in 72% of infected cells (p<0.001). Inside of the PV (lumen and parasites), [Ca++]i values appeared to be homogeneously distributed. At 48 hr after infection, the parasites had replicated and formed typical rosettes of more than 16 parasites. At this step, a positive [Ca++]i gradient (PV/cytoplasm) was detected in all analyzed cells (p<0.001). This result suggests that the PV (lumen and parasites) represents an individual subcellular compartment within the host cell that includes an independent [Ca++]i. Moreover, after 48 hr the cytoplasmic [Ca++]i decreased significantly (39 nM) compared with that measured from uninfected cells (53 nM) (p <0.05). Furthermore, the exit of Toxoplasma mediated by the calcium ionophore 4BrA23187 was preceded by a rise of [Ca++]i to 1 mM in the PV. The [Ca++]i rise and the liberation of parasites from their host appear to be correlated. On the basis of these observations, we suggest that the increase of [Ca++]i in the vacuole may act as a signal that triggers the egress of T. gondii.     

Engagement of human PECAM-1 (CD31) on human endothelial cells increases intracellular calcium ion concentration and stimulates prostacyclin release

Platelet-endothelial cell adhesion molecule-1 (PECAM-1) is a member of the immunoglobulin superfamily that plays a role in a number of endothelial cell (EC) functions including migration, angiogenesis, and transmigration of leukocytes across endothelium. We postulated that one way PECAM-1 might exert its effects was by regulating intracellular EC levels of calcium. Using single-cell fluorometry, we found that engagement of PECAM-1 by mAbs induced a slow but sustained increase in intracellular calcium, both in EC and in an adherent PECAM-1-transfected cell line that models endothelium. Generation of this signal was specific for certain anti-PECAM-1 antibodies, required the presence of the cytoplasmic domain, depended on extracellular calcium and on tyrosine phosphorylation, but did not require cross-linking; in fact, calcium increases were stimulated by certain Fab fragments. Activation of EC by PECAM-1 also caused a time-dependent increase in prostacyclin release. Given the importance of intracellular calcium and prostacyclin release as signaling molecules, engagement of PECAM-1 during cell-cell interactions may alter a number of EC functions including secretion of vasoactive mediators.     

High magnesium concentration inhibits ligand-stimulated calcium influx and hormone secretion in rat pituitary lactotropes with involvement of intracellular free magnesium

The effects of extracellular magnesium concentration ([Mg2+]ex) on thyrotropin-releasing hormone (TRH)-stimulated intracellular free calcium mobilization and prolactin secretion were investigated concomitantly with measurement of the intracellular free magnesium concentration ([Mg2+]i). TRH-stimulated intracellular free calcium mobilization was significantly inhibited when the medium was replaced by high Mg2+ medium ([Mg2+]ex = 10 mM) in normal Ca2+ medium. The inhibitory effects of high Mg2+ became apparent concomitantly with an increase in [Mg2+]i from 0.7 to 1.3 mM. High Mg2+ significantly inhibited TRH-induced PRL secretion in a dose-dependent manner in normal Ca2+ medium. TRH-stimulated inositol triphosphate (IP3) production was rather augmented by the replacement with high Mg2+ medium. In summary, high Mg2+ inhibits Ca2+ influx stimulated by TRH in the rat pituitary lactotropes, possibly with the involvement of [Mg2+]i increase. These results have general importance in relation to high Mg(2+)-induced suppression of the biological functions of cells.     

The effect of angiotensin II on platelet intracellular free magnesium and calcium ionic concentrations in essential hypertension

OBJECTIVE: To assess the effects of angiotensin II on intracellular free Mg2+ and Ca2+ concentrations in platelets from normotensive and hypertensive subjects. DESIGN AND METHODS: Seventeen normotensive, 25 untreated hypertensive and 18 treated hypertensive patients were studied. Intracellular Mg2+ concentrations were measured with the fluorescent dye mag-fura-2-acetyoxymethylester (AM) and intracellular Ca2+ concentrations with the fluorescent dye fura-2AM under basal conditions and after stimulation by angiotensin II, saralasin (angiotensin II antagonist), arginine vasopressin and endothelin-1. The effects of increased extracellular Mg2+ concentrations on intracellular Mg2+ and Ca2+ concentrations were also determined. RESULTS: The intracellular basal Ca2+ concentration was significantly higher in the untreated hypertensives compared with the normotensives and treated hypertensive subjects (150 +/- 14 nmol/l versus 120 +/- 17 nmol/l for normotensives and 124 +/- 8 nmol/l for treated hypertensives). The basal intracellular Mg2+ concentration was significantly lower in the untreated hypertensive compared to the normotensive and treated hypertensive groups (0.37 +/- 0.08 mumol/l versus 0.58 +/- 0.09 mumol/l for normotensives and 0.52 +/- 0.11 mumol/l for treated hypertensives). In the hypertensive groups, inverse correlations were found between intracellular Ca2+ and intracellular Mg2+ concentrations (r = -0.44, P < 0.05) and between intracellular Mg2+ and diastolic blood pressure (r = -0.35, P < 0.05), while a positive correlation was found between intracellular Ca2+ and systolic blood pressure (r = 0.41, P < 0.05). Exposure of the platelets to 1 nmol/l angiotensin II significantly increased intracellular Ca2+ and significantly decreased intracellular Mg2+ concentrations in all three groups. The angiotensin II-evoked effect on intracellular Ca2+ was exaggerated in the untreated hypertensives and blunted in the treated patients (basal versus stimulated: 150 +/- 14 versus 217 +/- 20 nmol/l in untreated hypertensives; 124 +/- 8 versus 140 +/- 10 nmol/l in treated hypertensives). Saralasin (0.1 mumol/l) abolished the effects of angiotensin. Arginine vasopressin (1 mumol/l) increased the intracellular Ca2+ concentration, whereas endothelin-1 (1 nmol/l) had no significant effect on either intracellular Ca2+ or intracellular Mg2+. Increasing extracellular Mg2+ concentrations led to significant reductions in intracellular Ca2+ concentrations in all groups and a significant elevation of the intracellular Mg2+ concentration in the untreated hypertensive patients only. CONCLUSIONS: These data demonstrate a relationship between angiotensin II and intracellular magnesium and calcium. In hypertension, angiotensin II-stimulated calcium responses may be related to simultaneously decreased intracellular magnesium concentrations.     

Modulation of intracellular calcium in human neutrophils by peripheral benzodiazepine receptor ligands

Persistent disabling groin pain in an active sportsman is a frustrating diagnostic and management problem for both the athlete and physician. After clinical examination and investigation there remains a group of patients who have unexplained groin pain, and may undergo lengthy periods of conservative management with numerous radiological investigations. Here we highlight such a case.     

Differential expression of thyroid hormone receptor isoforms in neurons and astroglial cells

The brain has abundant nuclear T3-binding sites and contains messenger RNAs (mRNAs) encoding multiple thyroid hormone receptor (TR) isoforms; the cellular distribution of these different TR isoforms is unknown. To determine whether the TR isoforms are differentially expressed in neuronal and astroglial cells, we examined the relative abundance of the mRNAs encoding TR alpha 1, c-erbA alpha 2, and TR beta 1 in primary cultures of fetal rat brain and in several cell lines of neural and glial origin. Additionally, the TR isoform polypeptides were identified by immunocytochemistry using isoform-specific antibodies. Northern blot analysis showed that fetal brain cell cultures contain mRNAs encoding the T3-binding isoforms TR alpha 1 and TR beta 1 as well as the mRNA encoding the non-T3-binding c-erbA alpha 2. c-erbA alpha 2 mRNA was most abundant, comprising more than 85% of the TR mRNAs in the primary cultures. Neuronal enrichment by antimitotic selection increased TR beta 1 mRNA approximately 3-fold, decreased c-erbA alpha 2 mRNA 70%, and had little or no effect on TR alpha 1 mRNA. Neuronal depletion resulted in the complete loss of TR beta 1 mRNA without changing c-erb alpha 2 or TR alpha 1 mRNA levels. Primary cultures of rat astrocytes, the astrocytoma cell line C6, and the pheochromocytoma cell line PC12 contained only the c-erbA alpha 2 mRNA. Immunocytochemistry using isoform-specific anti-sera revealed that TR beta 1 was exclusively localized to neuronal nuclei, and c-erbA alpha 2 was only found in the nuclei of astrocytes. These results show that TR beta 1 is localized to the nuclei of neuronal cells, and that c-erbA alpha 2 is restricted to the nuclei of astrocytes.     

Characterization of a nontumorigenic human breast epithelial cell line stably transfected with the human estrogen receptor (ER) cDNA

The accuracy of multichannel magnetocardiography (MCG) for the non-invasive localization of cardiac arrhythmias was investigated. A non-magnetic catheter was used in phantom studies and for cardiac pacing of 6 patients. In a clinical setting, 32 patients with WPW-syndrome, 37 patients with premature ventricular complexes and 12 patients with ventricular tachycardia were studied and the MCG results compared to reference methods, including invasive electrophysiological mapping. Phantom and pacing studies demonstrated the spatial localization accuracy to be better than 15 mm for a dipole-to-dewar distance below 15 cm. In all patients with structural cardiac disease, the ectopic focus was localized at the margin of the damaged area, serving as a proof of MCG localization. Invasive mapping confirmed the MCG result whenever performed (42 patients). In 11 patients (9 WPW, 2 VT) the MCG localization result was verified by successful HF catheter ablation as a gold standard. MCG permits the non-invasive localization of cardiac arrhythmias with high spatial accuracy. MCG guided HF catheter ablation constitutes a new concept of non-invasive localization and minimally invasive causal therapy.     

Human osteoclast-like cells are formed from peripheral blood mononuclear cells in a coculture with SaOS-2 cells transfected with the parathyroid hormone (PTH)/PTH-related protein receptor gene

Subclones of the human osteosarcoma cell line SaOS-2 were established by transfecting with an expression vector containing the human PTH/PTH-related protein (PTHrP) receptor, and their abilities to support osteoclast-like multinucleated cell (OCL) formation were examined in coculture with mouse or human hemopoietic cells. Of four subclones examined, SaOS-2/4 and SaOS-4/3 bound high levels of [125I]-PTH and produced a significant amount of cAMP in response to PTH. OCLs were formed in response to PTH in the cocultures of mouse bone marrow cells with either SaOS-2/4 cells or SaOS-4/3 cells. Human OCLs were also formed in response to PTH in the coculture of SaOS-4/3 cells and human peripheral blood mononuclear cells. Adding dexamethasone together with PTH greatly enhanced PTH-induced human OCL formation. Like mouse OCLs, human OCLs formed in response to PTH were tartrate-resistant acid phosphatase positive, expressed abundant calcitonin receptors and vitronectin receptors, and formed resorption pits on dentine slices. Other osteotropic factors such as 1alpha,25-dihydroxyvitamin D3, prostaglandin E2, and interleukin 6 plus soluble interleukin 6 receptors failed to induce mouse and human OCLs in cocultures with SaOS-4/3 cells. Both mouse and human OCL formation supported by SaOS-4/3 cells were inhibited by either adding an antibody against macrophage-colony stimulating factor or adding granulocyte/macrophage-colony stimulating factor. Thus, it is likely that human and mouse OCL formation supported by SaOS-4/3 cells are similarly regulated. These results indicate that the target cells of PTH for inducing osteoclast formation are osteoblast/stromal cells but not osteoclast progenitor cells in the coculture. This coculture model will be useful for investigating the abnormalities ofosteoclast differentiation and function in human metabolic bone diseases.     

Differential expression of thrombospondin, collagen, and thyroglobulin by thyroid-stimulating hormone and tumor-promoting phorbol ester in cultured porcine thyroid cells

In the present study, we have investigated the potential regulation of thyroglobulin (Tg) and extracellular matrix components synthesis by thyroid-stimulating hormone (TSH) and tetradecanoyl phorbol-13-acetate (TPA) on thyroid cells. Porcine thyroid cells isolated by trypsin-EGTA digestion of thyroid glands were maintained in serum containing medium on poly (L-lysine)-coated dishes. Cells differentiated into follicular or vesicular-like structures were distinguished by their ability to organify Na[125I] and to respond to TSH stimulation. After an incubation of the cells with radiolabeled proline or methionine, two major proteins were identified, p450-480 and p290 (so named because of their molecular masses). Tg (p290) synthesis was demonstrated by the synthesis of [131I]-labeled polypeptides with electrophoretic properties identical to those of authentic Tg molecules. P450-480 resolved to M(r) 190,000 under reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) conditions. It was identified as thrombospondin by its reactivity with a monoclonal anti-human thrombospondin and by peptide sequencing of some of its tryptic fragments that displayed identity to thrombospondin I. Collagen synthesis was demonstrated by the formation of radioactive hydroxyproline and by the synthesis of pepsin-resistant polypeptides ranging from M(rs) 120,000 to 200,000. When the cells were cultured in the presence of 100 nM TPA, the culture medium contents of thrombospondin and collagen were increased by 2.7 and 1.6-fold, respectively, whereas Tg content was decreased by a factor 3.9. In contrast, the acute treatment of control cells with TPA induced a decrease in both Tg and collagen content by factors 3.0 and 1.5, respectively, and an increase in thrombospondin content by a factor 2.5. In the presence of 100 nM TPA, TSH (1 mU/ml) did not counteract the stimulating effect of TPA on extracellular matrix components synthesis. In contrast, when cells were cultured in the presence of TSH alone at concentrations higher than 0.1 mU/ml, collagen and thrombospondin in the medium were decreased by a factor 2.0 and 1.9, respectively, and TSH preferentially activated Tg synthesis. However, no acute response to TSH was observed in cells incubated for 2 days without effectors (control cells). On TSH differentiated cells, TPA decreased both collagen and Tg accumulation by factors 1.2 and 1.8, respectively, whereas it increased the one of thrombospondin by a factor 2. These results, together with the stimulating effect of TPA on TSH mediated cell proliferation, argue for a role of thrombospondin in cell adhesion and migration events within the thyroid epithelium.     

Effects of erythrocyte lysate of different incubation times on intracellular free calcium in rat basilar artery smooth-muscle cells

The effects of 5-hydroxytryptamine (5-HT) on ethanol-induced gastric mucosal damage and on epithelial and vascular integrity were investigated. Male Sprague-Dawley rats were administered with 5-HT (5 or 10 mg/kg, IP) 30 min prior to the challenge with ethanol (40% v/v, 10 ml/kg, PO). 5-HT dose dependently aggravated ethanol-induced injury in the gastric mucosa. Both xanthine oxidase (XO) and myeloperoxidase (MPO) activities in the mucosa were significantly increased with the high dose of 5-HT, which also potentiated the elevation of these enzyme activities by ethanol. However, the mucosal superoxide dismutase activity was left unaltered. In neutropenic (antineutrophil serum-treated) animals, the ethanol-induced gastric mucosal injury was significantly ameliorated, with or without the pretreatment of 5-HT (10 mg/kg). In addition, the effect of 5-HT on the activity of MPO, but not of XO, was also attenuated in these animals. In the ex vivo gastric chamber study on pentobarbital-anesthetized animals, volume of gastric secretion was significantly decreased in the 5-HT-treated groups, with further reduction after ethanol incubation. Transmucosal potential difference (PD) was significantly reduced in 5-HT-treated rats, which also potentiated the ethanol-induced drop in PD. Nevertheless, 5-HT dose dependently increased mucosal vascular permeability and further enhanced during ethanol incubation. These findings suggest that 5-HT adversely affects the defense mechanisms of the gastric mucosa by reducing the secretory function of the mucosal cells and to weaken the epithelial and vascular integrity. Neutrophil activation appears to be responsible for the detrimental effects of 5-HT partly through the elevation in MPO activity. The increase in mucosal XO activity by 5-HT may induce free radical production and possibly modulate the ulcerogenic processes.