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Biomedical subjects

C M Rotella

Publications and source records attributed to C M Rotella.

At least 73 records · Page 4Linked to original sources
PubMed1994

Acetaldehyde induces c-fos and c-jun proto-oncogenes in fat-storing cell cultures through protein kinase C activation.

Hepatic fibrosis is an important morphological feature of alcohol-induced liver injury. We previously reported that acetaldehyde stimulates collagen I and fibronectin gene transcription in rat fat-storing cell (FSC) culture. We here evaluated whether acetaldehyde increases Col I and FN gene transcription through the induction of c-fos and c-jun proto-oncogenes and studied the possible role played by protein kinase C (PKC) and c-AMP. FSCs, isolated from rat liver on a Nycodenz density gradient, were exposed to acetaldehyde for 1/2, 1, 3, 6, 12, 24 hr and for 10, 20, 30, 45, 60, 90 min in the experiments for jun and fos expression, respectively. Acetaldehyde produced a rapid and transient induction of fos mRNA (undetectable at t = 0, peak at t = 45 and still evident at t = 90). Jun mRNA was weakly expressed in unstimulated FSCs; acetaldehyde induced a prolonged activation of jun expression up to 24 hr with a peak at 3 hr. To study the role of PKC were repeated the experiments in the presence of Staurosporine and H-7. These inhibitors of PKC activity blocked the stimulatory effect of acetaldehyde on fos and jun mRNA expression. Furthermore, they abolished the stimulatory effect of acetaldehyde on collagen I and fibronectin gene expression by FSCs. Acetaldehyde increased the cell membrane PKC activity in FSC cultures in a dose-dependent way. Intracellular cAMP levels were not significantly modified by acetaldehyde in the first 30 min of incubation. We conclude that acetaldehyde increases procollagen I and fibronectin gene transcription in FSCs, possibly through c-fos and c-jun expression, and that PKC may play a regulatory role in this chain of events.

Acetaldehyde↗
PubMed1993

Release of extracellular matrix components by bovine bone endothelial cells in continuous culture.

Production of collagen type I (pIp), type III (pIIIp), type IV (CIV), type VI (CVI), fibronectin (FN), laminin and glycosaminoglycans (GAGs) has been investigated in a clonal line of Bovine Bone Endothelial (BBE) cells continuously growing in culture. BBE cells produced pIp, pIIIp and FN, whereas they did not synthetize significant amounts of CIV, CVI and laminin. pIp, pIIIp and FN syntheses were influenced by cell density:pIp secretory activity reached its maximum just after cell seeding, whereas pIIIp and FN production increased later, when cells were subconfluent. Furthermore, BBE cells produced and released significant amounts of GAGs into medium.

Animals↗
PubMed1993

Regulation of extracellular matrix synthesis by transforming growth factor beta 1 in human fat-storing cells.

BACKGROUND: Fat storing cells (FSC) are nonparenchymal liver cells generally considered the major source of the hepatic extracellular matrix (ECM). Transforming growth factor beta 1 (TGF-beta 1) is a potent regulator of ECM synthesis in various cell types. In this study, the effect of TGF-beta 1 on procollagen types I, III, IV, laminin (Lam), and fibronectin (FN) synthesis in cultured human FSCs was analyzed. METHODS: FSCs were isolated from wedge sections of normal human livers. Morphological studies were performed by immunofluorescence and electron microscopy. ECM components in human FSC cultures were measured by an enzyme-linked immunosorbent assay. The expression of messenger RNA (mRNA) was evaluated by Northern blot and in situ hybridization. RESULTS: Cultured human FSCs displayed numerous fat droplets in the perinuclear zone, and immunoreactivity for vimentin and alpha-smooth muscle actin. A weak nonfibrillar staining was observed by using a polyclonal antidesmin antibody. TGF-beta 1 induced a dose-dependent increase of procollagen I, III, and FN accumulation in human FSC cultures, whereas procollagen IV and Lam production was not affected. Furthermore, TGF-beta 1 increased the expression of alpha 1 (I), alpha 1 (III) procollagen, FN and TGF-beta 1 mRNA in human FSC cultures. CONCLUSIONS: These data indicate that TGF-beta 1 is able to increase the synthesis of procollagen I, III, and FN in cultured human FSCs. Moreover, TGF-beta 1 can induce its own mRNA in the same cells.

Adipose Tissue↗
PubMed1993

Acetaldehyde-protein adducts, but not lactate and pyruvate, stimulate gene transcription of collagen and fibronectin in hepatic fat-storing cells.

Hepatic fibrosis is an important morphological feature of alcohol-induced liver injury. We previously reported that acetaldehyde, but not ethanol can stimulate type I collagen and fibronectin synthesis in cultures of rat fat-storing cells (FSC) by increasing transcription of the specific genes. The effect of lactate and pyruvate was studied on collagen I, III, fibronectin accumulation by cultured rat FSCs and it was investigated whether acetaldehyde could increase procollagen I and fibronectin gene transcription through the formation of protein adducts. Lactate and pyruvate (5, 15 and 25 mmol/l) did not significantly affect collagen I, III and fibronectin production by cultured FSCs. Pyridoxal-phosphate and p-hydroxymecuribenzoate (inhibitors of acetaldehyde-protein adduct formation) blocked the stimulatory effect of acetaldehyde on procollagen I and fibronectin gene transcription. These data suggest that ethanol may act as a liver fibrogenic factor through acetaldehyde, its immediate metabolite, whereas lactate does not seem to play a role. Acetaldehyde might stimulate gene transcription of extracellular matrix components by liver FSCs through the formation of adducts with proteins.

Acetaldehyde↗
PubMed1991

Calcitonin gene-related peptide increases the production of glycosaminoglycans but not of collagen type I and III in cultures of rat fat-storing cells.

We investigated whether calcitonin gene-related peptide (CGRP) was able to affect the production of collagen and glycosaminoglycans (GAG) in cultures of rat fat-storing cells (FSC). Rat CGRP (1 nM-1 microM) induced a dose-dependent increase of total GAG production in FSC cultures with an EC50 of 28 nM. One uM human CGRP (8-37) shifted the dose-response curve of rat CGRP to the right (EC50 = 257 nM) without depressing the maximal response. Salmon calcitonin (1 nM-1 microM) did not significantly modify total GAG accumulation in FSC cultures. Collagen type I and III production was not significantly affected by either CGRP or calcitonin in FSC cultures. These findings suggest that peripheral sensory neuropeptides may modulate liver fibrogenesis.

Adipose Tissue↗
PubMed1990

Intrathyroidal lymphocytes from non toxic multinodular goiter: no evidence for production of thyroid stimulating antibodies.

Although an autoimmune pathogenesis for non toxic goiter has been suggested, reports concerning circulating antibodies to TSH receptor structures have been conflicting. Intra thyroid lymphocytes, capable of secreting IgG, have been shown to be involved in the pathogenesis of Graves' and Hashimoto's diseases; therefore, the ability of conditioned media obtained from intra thyroid lymphocyte culture, and of IgG purified from these media, to stimulate cAMP accumulation and [3H]-Thymidine (TdR) uptake in FRTL-5 cells was investigated. The activity of IgG produced "in vitro" was compared with that of circulating IgG. Thyroid tissue samples were obtained at surgery from 21 patients with non toxic multinodular goiter (MNG), 5 patients with active Graves' disease (GD), and from 10 normal subjects, undergoing neck surgery for non-thyroidal pathology. IgG purified from media of GD lymphocyte cultures stimulated both cAMP accumulation and [3H]-TdR in 5 out of 5 cases: all of the IgG purified from control or MNG lymphocyte culture media was not active in either assay. Circulating IgG did not affect cAMP accumulation or [3H]-TdR in any of the non toxic MNG cases: controls showed no changed at all. However, both activities represented were increased by GD IgG. Conditioned media from intra thyroid lymphocyte cultures significantly inhibited basal cAMP accumulation in 7 out of the 21 non toxic MNG samples and totally abolished the response in all GD patients. [3H]-TdR was not affected by IgG of any of the controls, but it had an inhibitory effect on 8 out of 21 non toxic MNG patients, and significantly stimulated [3H]-TdR in all GD patients. In conclusion, present data demonstrate that intra thyroid lymphocytes from non toxic MNG do not produce antibodies capable of mimicking TSH actions through the adenylate cyclase cascade. Conversely, soluble factors interacting in TSH-mediated functions of FRTL-5 cells are present in conditioned media of intra thyroid lymphocytes of GD and MNG thyroid lymphocytes of GD and MNG thyroid cultures.

Adolescent↗
PubMed1989

Characterization of receptors for insulin and insulin-like growth factor-1 on FRTL-5 thyroid cells.

FRTL-5 rat thyroid cells have receptors for both insulin and IGF-I which can be distinguished in binding studies. The ability of TSH to regulate each in an antiparallel manner is atypical. If these receptors are shown to have independent as well as coordinate activities, studies of the mechanisms of their receptor cross-talk in these cells will be relevant to understanding IGF-I and insulin receptors in other tissues.

Animals↗
PubMed1989

Receptors of the thyroid: the thyrotropin receptor is only the first violinist of a symphony orchestra.

A basic reason for undertaking these studies was to further our knowledge of the structure and function of the TSH receptor as well as its interaction with other receptors on thyroid cells. The multiplicity of observations suggests the approach is bearing fruit, does not provide a simple answer, and can have pitfalls. We hope they may also contribute to understanding the structure and function of autoantigens in Graves' disease and glycoprotein hormone receptors in general. The authors are grateful to their collaborators in the National Dental Institute, particularly Drs. Bellur Prabhakar, Edward Oates, and Abner Notkins, in the National Cancer Institute, Drs. W. O. McBride and M. Lerman for their contributions to the cloning studies.

Amino Acid Sequence↗
PubMed1989

Calf serum modifies the mitogenic activity of epidermal growth factor in WRT thyroid cells.

It has already been shown that Wistar rat thyroid (WRT) cells in low concentrations of calf serum (0.5%) are under the influence of both thyrotropin (TSH) and insulin as regards growth. The present data show that epidermal growth factor (EGF), in concentrations up to 10 micrograms/ml, is not able to modify DNA synthesis in WRT cells. On the other hand, insulin-like growth factor I (IGF-I) stimulates DNA synthesis from a dose which is 10-fold lower than that of insulin alone. Combined stimulation of EGF and TSH in WRT cells is equal to that of TSH alone in relation to DNA synthesis, while the combined presence of TSH and IGF-I, or TSH and insulin, in the same medium results in an effect which is greatly superior to the theoretical sum of activities. Repetition of the same experiments using the original clone of WRT cells, but in high concentrations of calf serum (5%), shows that EGF stimulates DNA synthesis in a dose-dependent way from 0.1 to 100 ng/ml. Under these conditions, combined stimulation of EGF with TSH shows that DNA synthesis is equal to the predicted theoretical sum. No other differences in WRT cell sensitivity to either IGF-I or insulin, or IGF-I and TSH and insulin and TSH, can be noted. This finding is confirmed by the demonstration of specific and sensitive binding sites for EGF on WRT cells cultured in 5% calf serum; these binding sites are not present on WRT cells adapted to grow in 0.5% calf serum. Present data support the hypothesis that EGF and serum growth actions are mediated through the same analogous pathway, which is, however, different from those of TSH and/or IGF-I and/or insulin.

Animals↗
PubMed1989

Insulin-like growth factor-I: autocrine secretion by human thyroid follicular cells in primary culture.

Insulin-like growth factor-I (IGF-I) stimulates the growth of thyroid cells of different animal species. However, conflicting results have been reported as regards the function and mechanism of the action of IGF-I at the thyroid level. This study was designed to determine whether normal human thyroid cells have IGF-I receptors and whether IGF-I could act on such cells through an autocrine mechanism. Human thyroid follicular cells in primary culture, not contaminated by fibroblasts, were used. They had specific and saturable binding sites for IGF-I, as revealed by radiolabeled binding method, and displayed an average receptor number of 2000/cell. Under the same experimental conditions, insulin receptors were not detectable. Human thyroid follicular cells secreted IGF-I into the culture medium, as assessed by a specific chemiluminescent immunoassay. The IGF-I secretory process was detectable for at least 12 days of culture, but a high degree of variability has been found among individual samples. Acid-gel chromatography demonstrated that IGF-I and a higher mol wt IGF-I, most likely IGF-I bound to its binding protein, were secreted by human thyroid cells. TSH stimulated the secretion of the two molecules in normal human thyroid cells. The TSH effect on IGF-I secretion was concentration dependent between 0.1 nmol/L and 0.1 mumol/L. GH stimulated IGF-I synthesis by thyroid cells in a concentration range from 20-200 micrograms/mL. Since binding studies demonstrated the presence of IGF-I receptors on human thyroid cells, IGF-I probably regulates human thyroid function through an autocrine mechanism.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1989

Regulation of amino acid transport in rat and human thyroid cells.

To clarify the role of insulin, IGF-I and TSH in thyroid cell regulation, their effects on amino acid transport were studied separately. These effects were noted and compared using both the Wistar rat thyroid cell line and human thyroid cell cultures. Insulin, IGF-I and TSH were able independently to induce the radiolabelled alpha-aminoisobutyric acid transport within the rat thyroid cells: TSH stimulated the amino acid transport in rat thyroid cells in a dose-dependent way from 1 pmol/l to 10 nmol/l. Similarly, insulin increased amino acid transport significantly from 0.17 nmol/l up to 0.17 mumol/l and IGF-I from 0.13 pmol/l up to 0.13 mumol/l. The combined effects of insulin and TSH on amino acid transport were equal to the theoretical sum of the activities, whereas those of IGF-I and TSH were greater than the theoretical one. When human thyroid cell cultures were used, a significant increase of labelled amino acid transport was induced by TSH, i.e. from 0.1 pmol/l to 10 pmol/l; IGF-I stimulated amino acid transport in a range from 0.13 pmol/l to 13 pmol/l, under the same conditions. Conversely, only large doses of insulin, i.e. 1.7 nmol/l, were able weakly to stimulate amino acid transport. When submaximal TSH and IGF-I doses were co-incubated in human thyroid cells, an additive effect on amino acid transport was observed.

Adult↗
PubMed1987

Cholinergic control of cyclic nucleotide metabolism in human thyroid cells.

In the presence of Ro 20-1724, a selective inhibitor of cyclic nucleotide phosphodiesterase, carbamylcholine increases cAMP and cGMP levels in human thyroid cells in primary culture. The increase of cAMP exhibited at concentrations of carbamylcholine between 10 fM and 10 pM, is dose- and time-dependent, it is maximum after 30 min and is abolished after 60 min. At higher carbamylcholine concentration (10 microM), cAMP increases rapidly, becoming maximum after 15 min, but returns to unstimulated values after 30 min. The increase of cGMP is also dose-dependent (0.1 nM-10 microM); it reaches the maximum after 30 min and returns to unstimulated values after 120 min. A significant increase of phosphodiesterase activity is observed at 10 microM carbamylcholine. Atropine, a muscarinic receptor antagonist, blocks carbamylcholine effects on both cAMP and cGMP production without affecting the thyrotropin-induced cAMP accumulation. Hexamethonium, a nicotinic receptor antagonist does not affect the cholinergic effects. In the presence of Ro 20-1724, 10 microM carbamylcholine significantly inhibits the effect of thyrotropin on cAMP production, while the combined addition of low doses of carbamylcholine and thyrotropin (0.1 nM and 10 pM, respectively) results in an additive effect on cAMP levels. Inhibition of thyrotropin activity on cAMP production, similar to that exerted by 10 microM carbamylcholine is produced by increasing free intracellular calcium; this inhibition is relieved by using a calmodulin-sensitive phosphodiesterase inhibitor, M and B 22948 at 50 microM dose. High concentrations (10 microM) of carbamylcholine increase the adenylate cyclase activity, without any significant effect on the thyrotropin-induced activation of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗
PubMed1987

Insulin stimulates cell growth of a new strain of differentiated rat thyroid cells.

A new strain, named WRT cells, has been generated from primary cultures of rat thyroids. The primary culture was grown in Coon's modified Ham's F12 medium with 5% calf serum, insulin, hydrocortisone, transferrin, somatostatin, glycyl-L-histidyl-L-lysine and thyrotropin (TSH). On the basis of the following facts, the WRT cell strain, cloned from the primary culture, was considered 'normal': the cells are euploid, not carcinogenic, not able to grow in soft agar, and show contact inhibition. Their differentiated functions consist of the ability to synthesize thyroglobulin and to take up iodide, and they have a TSH-dependent adenylate cyclase system. TSH increases cellular adenosine 3',5'-cyclic monophosphate (cAMP) levels and [3H]thymidine incorporation in WRT cells from a concentration similar to that active on another clonal rat cell line (FRTL-5), even though the cell replication appears to be differently regulated in the two cell strains. In fact, the WRT cell doubling time is 42 h and they are also able to grow in the absence of TSH, though more slowly. In the same conditions, FRTL-5 cells have a population doubling time of 38 h, but they are not able to grow in the absence of TSH. When the effect of the other growth factors of the medium was studied, insulin appears to be a growth stimulus by itself, while it is only a facilitative step for TSH action in FRTL-5 cells. WRT cells, unlike FRTL-5 cells, can grow with a population doubling time of 80 h, when cultured for prolonged periods in a medium with a low serum concentration (0.5%), but containing insulin plus TSH. In conclusion, the WRT cell strain is a new and interesting experimental model for studying growth factors at the level of the thyroid, especially for their mechanism of action on the TSH receptor.

Animals↗
PubMed1986

Thyrotropin-independent mutant clones from FRTL5 rat thyroid cells: hormonal control mechanisms in differentiated cells.

Mutant cells varying in the pathways of their responses to hormonal stimulation are useful in defining the subcellular steps in the mechanisms of hormone action. FRTL5, a strain of normal and differentiated cells originally derived from adult rat thyroids, which depends on TSH for growth in vitro, was used to produce five TSH-independent mutants, after chemical mutagenesis and selection in medium lacking TSH. Their characterization and comparison with wild type cells demonstrated full retention of differentiated thyroid function markers such as thyroglobulin production and active iodide transport, and a slower growth rate. Characterization of cAMP metabolism in mutants revealed levels of basal cAMP and adenylate cyclase and phosphodiesterase activities similar to those of wild type cells kept in a nonproliferative state in medium lacking TSH. Adenylate cyclase responsiveness to very low doses of TSH (10(-12) M) was fully retained in all mutant clones, but the TSH-dependent cAMP elevation, although comparable to that reported in wild type cells, was not followed by significant growth stimulation in mutants. These findings demonstrate that the persistence of functional TSH receptors in these cells and that of growth regulation in them is independent of cAMP elevation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1986

Ability of monoclonal antibodies to the thyrotropin receptor to increase collagen synthesis in human fibroblasts: an assay which appears to measure exophthalmogenic immunoglobulins in Graves' sera.

Immunoglobulin G (IgG) preparations from 17 of 20 hyperthyroid patients with Graves' ophthalmopathy stimulated collagen biosynthesis in human fibroblasts, as measured by [3H]proline incorporation. This activity was not associated with thyroid-stimulating antibody (TSAb) activity in a thyroid cell cAMP assay in 50% of the IgG preparations, and it was not found in IgGs from 12 normal subjects, 7 of 8 patients with Graves' hyperthyroidism but no ophthalmopathy, 4 patients with Hashimoto's disease, 7 patients with nontoxic goiter, or 4 hypothyroid patients. In the same assay, 11E8, 22A6, and 13D11, 3 mouse monoclonal antibodies to the bovine TSH receptor, and 307H6, a human monoclonal antibody to the TSH receptor of the thyroid from a Graves' patient with ophthalmopathy, also stimulated [3H]proline incorporation into collagen and were active at more than 1,000- to 10,000-fold lower IgG concentrations (0.1-0.5 microgram/ml as opposed to greater than 1 mg/ml). 11E8 and 13D11 are TSH binding inhibitory antibodies (TBIAbs); 22A6 and 307H6 are TSAbs in cAMP assays. Two other mouse anti-TSH receptor monoclonal antibodies, both TBIAbs, as well as 8 human monoclonal antibodies to the TSH receptor from Graves' patients with or without ophthalmopathy (2 TBIAbs and 6 TSAbs) were negative or significantly less potent (greater than 50 fold) in the assay. The fibroblast activity of the monoclonal antibodies was lost if the antibodies were preincubated with thyroid membranes, was significantly decreased when fibroblasts were exposed to mild trypsin treatment before the assay, was not inhibited by human asialoagalacto-thyroglobulin, and required more than a TSH receptor determinant, since TSH alone neither duplicated nor inhibited the antibody activity. In summary, an assay for measuring the activity of autoantibodies active in causing ophthalmopathy is described, and some but not all TSH receptor monoclonal antibodies have been found to duplicate the action of the autoimmune IgGs from the ophthalmopathy patients.

Adult↗
PubMed1986

Unusual antagonistic actions of mixtures of vasoactive intestinal peptide, thyroid-stimulating antibodies, and cholera toxin on adenosine 3',5'-monophosphate accumulation in normal human thyroid cell cultures.

When individually tested, vasoactive intestinal peptide (VIP), cholera toxin, and monoclonal thyroid-stimulating antibodies (TSAbs), but not TSH binding-inhibiting antibodies (TBIAbs), elevate cAMP levels in cultured human thyroid cells. In this study, we tested the effect on cAMP levels in human thyroid cells of the simultaneous presence of VIP and the other ligands noted. Monoclonal TBIAbs (11E8 and 122G3), which interact with a membrane glycoprotein containing the high affinity binding site of the TSH receptor, did not alter VIP-induced cAMP accumulation in the thyroid cells. These data indicate that VIP and TSH bind to distinct sites on the cell membrane. In contrast, monoclonal TSAbs 208F7 and 307H6, which interact with a portion of the TSH receptor other than its high affinity binding site, not only did not have their usual agonist activity, but, instead, caused a marked inhibition of human thyroid cAMP accumulation when VIP was also present. Mutual antagonism by two agonists, i.e. inhibition evident when TSAbs and VIP were mixed, was also found when cholera toxin was coincubated with VIP. The inhibitory effect of mixing cholera toxin and VIP was nearly immediate and was duplicated with mixtures of the beta-subunit of cholera toxin and VIP. The inhibition evident in mixing VIP and the TSAbs or cholera toxin was not duplicated in mixtures of isoproterenol and VIP or in mixtures of forskolin and VIP. These results suggest that the mutual inhibition of VIP and either TSAbs or cholera toxin is at a step that couples the TSH and VIP high affinity receptor-binding sites to the adenylate cyclase complex.

Antibodies, Monoclonal↗