Biomedical subjects

M Saji

Publications and source records attributed to M Saji.

At least 109 records · Page 6Linked to original sources

PubMed1990

Evidence that glutamate is the transmitter mediating respiratory drive from medullary premotor neurons to phrenic motoneurons: a double labeling study in the rat.

Glutamate immunoreactivity was found in 81-84% of the bulbospinal neurons projecting to the phrenic motoneuron pool, which were located in the para-ambiguous nucleus and the ventral vicinity of the retrofacial nucleus. Their glutamate contents were identified by a double labeling technique in combination with the retrograde transport of horseradish peroxidase and immunocytochemistry of the glutamate. The remaining 16-19% of the bulbospinal neurons projecting to the phrenic motoneuron pool did not contain glutamate immunoreactivity. Thus it appears that glutamate may be a major transmitter of the inspiratory drive from the medullary respiratory neurons to the phrenic motoneurons.

Animals↗

PubMed1990

A microsequencing approach to identify proteins which appear to interact with thyrotropin in rat FRTL-5 thyroid cells.

In order to resolve questions concerning the in situ structure of the thyrotropin (TSH) receptor, [35S]methionine-labeled thyroid cell preparations were detergent solubilized and proteins exhibiting TSH-dependent binding to TSH-Sepharose were identified. Two such proteins, 43 and 70 kd, are identified in this report as gamma-actin and a member of the heat shock 70 protein family, respectively, based on the microsequence of two peptides from each. Identification of the former was confirmed by Western blotting and immunostaining using anti-actin, the latter by its ability to bind [32P]ATP, a characteristic feature of this family of proteins. The results suggest that TSH-cross linking reports defining TSH receptor subunits should be viewed with caution in the absence of comparative sequence data; consideration must, however, be given to the existence of receptor associated proteins.

Actins↗

PubMed1990

Thoracic expiratory motor neurons of the rat: localization and sites of origin of their premotor neurons.

The expiratory motor neurons of the representative thoracic segment of the rat were examined as to their localization and sites of origin of their premotor neurons in the lower brainstem. Either T6 or T7 was selected as a representative target segment for horseradish peroxidase (HRP) injection. The intercostal motor neurons in the T6 or T7 were doubly labeled by HRP placed in the cut end of the internal intercostal nerve and True blue placed in the cut end of the external intercostal nerve. The thoracic expiratory motor neurons labeled by HRP were concentrated in the oblique zone running along the dorsal to ventrolateral direction in both the T6 and T7 segments. By contrast, the thoracic inspiratory motor neurons labeled by True blue were concentrated in the horizontal zone running along the bottom of the ventral horn in both the T6 and T7 segments. A small amount of HRP was iontophoretically injected through a double-barrel coaxial electrode to sites of the expiratory motor neurons which had been identified electrophysiologically. In 5 successful experiments, the HRP-labeled cells were bilaterally distributed in the para-ambiguus nucleus (59.0%), ventral subnucleus of the paramedian reticular nucleus (13.6%), and raphe nuclei (10.8%). In another experiment, it was found that the expiratory premotor neurons in the para-ambiguus nucleus were present in a narrow column over the entire length of the nucleus at a level between 1.0 mm rostral and 1.4 mm caudal to the obex.

Animals↗

PubMed1990

Cloning, chromosomal assignment, and regulation of the rat thyrotropin receptor: expression of the gene is regulated by thyrotropin, agents that increase cAMP levels, and thyroid autoantibodies.

A rat thyrotropin (thyroid-stimulating hormone, TSH) receptor cDNA was isolated that encoded a protein of 764 amino acids, Mr 86,528. Transfection of the cDNA caused COS-7 cells to develop a TSH-sensitive adenylate cyclase response and the ability to bind 125I-labeled TSH; both activities were similar to those of rat FRTL-5 thyroid cells and not duplicated by lutropin. The gene represented by the cDNA was assigned to mouse chromosome 12 and human chromosome 14. Northern analyses identified two species of mRNA, 5.6 and 3.3 kilobases, in FRTL-5 thyroid cells; the transcripts appeared to differ only in the extent of their 3' noncoding sequences. There were minimal amounts of the two mRNAs in rat ovary, and neither was detected in RNA preparations from rat testis, liver, lung, brain, spleen, and FRT thyroid cells, which do not have a functional TSH receptor. TSH decreased both mRNA species 3- to 4-fold within 8 hr in FRTL-5 thyroid cells; down-regulation was dependent on TSH concentration and duplicated by forskolin, cholera toxin, or 8-bromo-cAMP but not by a phorbol ester. Down-regulation was also duplicated by thyroid-stimulating autoantibodies, which increased cAMP levels, but not by thyrotropin binding-inhibiting auto-antibodies, which actually increased TSH receptor mRNA levels.

Amino Acid Sequence↗

PubMed1990

Effect of lithium on deoxyribonucleic acid synthesis and iodide uptake in porcine thyroid cells in culture.

We investigated the direct effect of lithium on porcine thyroid cells in culture to exclude the secondary regulatory factors. First we have studied the effect of lithium on TSH-induced iodide uptake. Significant suppression was seen at 0.1 mmol/liter, and half-maximal suppression was obtained at the pharmacological concentration reported in patient serum. The suppression was dose dependent and reversible. Besides the suppression of cAMP production stimulated by TSH, lithium also inhibited iodide uptake stimulated by forskolin or 8-bromo-cAMP. These results demonstrated that lithium inhibits TSH-induced iodide uptake not only by reducing cAMP production, but also by acting on the steps of post-cAMP production. Next, we studied the effect of lithium on DNA synthesis of the cultured porcine thyroid cells. Lithium stimulated [3H]thymidine incorporation of the thyroid cells in the basal condition (0.5% fetal calf serum) as well as those stimulated by insulin-like growth factor-I (100 micrograms/liter). The minimal concentrations for the significant increase were 0.5 and 0.1 mmol/liter, respectively. These results suggest that lithium might contribute to the formation of the goiter directly at the cellular levels in patients treated with the agent.

Animals↗

PubMed1990

Repeatedly passed FRTL-5 rat thyroid cells can develop insulin and insulin-like growth factor-I-sensitive cyclooxygenase and prostaglandin E2 isomerase-like activities together with altered basal and thyrotropin-responsive thymidine incorporation into DNA.

Repeatedly passed or aged rat FRTL-5 thyroid cells develop a high level of basal [3H]thymidine incorporation into DNA and a reduced response to TSH in medium containing 5% serum and insulin (5H medium). The basal [3H]thymidine incorporation into DNA of aged cells can exceed the TSH-induced increase in earlier passages of the same cell line (fresh cells) and the TSH response decreases from more than 10-fold above basal in fresh cells to less than 2-fold in aged cells. This change is not associated with a loss of the diploid karyotype, a change in basal cAMP levels, or a change in dependence on TSH for cell growth. Attenuation of the TSH response in the [3H]thymidine incorporation assay is more evident than the reduced effect of TSH on cAMP levels or iodide transport; moreover, the TSH effect on cAMP levels does not correlate with that on [3H] thymidine incorporation as a function of hormone concentration. The high basal activity in [3H]thymidine incorporation into DNA in aged cells is due to an increased responsiveness to insulin, insulin-like growth factor-I (IGF-I), or serum. Thus, removal of serum and insulin from the medium eliminates the high basal [3H]thymidine incorporation into DNA, and this activity is restored by insulin or IGF-I in a concentration-dependent manner. The increased responsiveness of aged cells to insulin or IGF-I is inhibited by indomethacin or hydrocortisone and is associated with insulin or IGF-I, but not TSH, stimulation of cyclooxygenase and prostaglandin E2 (PGE2) isomerase-like activity. Fresh cells, in contrast, require TSH plus insulin or IGF-I to increase these activities. Increased responsiveness of cyclooxygenase activity to insulin or IGF-I in aged cells reflects at least in part an increase in cyclooxygenase mRNA levels. We suggest that insulin/IGF-I stimulation of PGE2 production leads to the high basal thymidine incorporation into DNA in aged cells maintained in TSH-depleted (5H) medium; the reduced stimulation by TSH of cAMP content or iodide uptake may reflect PG inhibition (negative feedback regulation) of cAMP production.

Animals↗

PubMed1990

Effect of hydrocortisone on the ability of thyrotropin to increase deoxyribonucleic acid synthesis and iodide uptake in FRTL-5 rat thyroid cells: opposite regulation of adenosine 3',5'-monophosphate signal action.

In FRTL-5 rat thyroid cells, hydrocortisone alters two TSH-increased cAMP-mediated activities in an opposite manner. Thus, in a concentration-dependent fashion, hydrocortisone synergistically enhances TSH-increased thymidine incorporation into DNA, whereas it inhibits TSH-induced iodide uptake. The effect of hydrocortisone on TSH-increased thymidine incorporation is specific, in that it has only a minimal ability by itself to increase thymidine incorporation into DNA and slightly inhibits the activity of insulin or insulin-like growth factor-I. The effect of hydrocortisone on TSH-induced iodide uptake does not result from altered iodide efflux, but, rather, from a decrease in the maximal velocity, not the Km, of iodide influx, i.e. from a decrease in the effective number of iodide porters. The action of a cAMP analog to increase thymidine incorporation into DNA or iodide uptake was also increased or decreased, respectively, by hydrocortisone, whereas hydrocortisone did not diminish the ability of TSH to increase cAMP levels in the cells. The different effect of hydrocortisone on these two cAMP-mediated activities reflects, therefore, regulation of cAMP signal action rather than the ability of TSH to generate a cAMP signal. Twenty-four hours after TSH, actinomycin-D superinduces iodide uptake and abolishes the action of hydrocortisone to inhibit iodide uptake. It has been suggested that actinomycin-D superinduces iodide uptake in FRTL-5 cells by a posttranscriptional action, inhibition of mRNA degradation, rather than by its known transcriptional actions linked to DNA synthesis. More recent studies of the effect of actinomycin-D, given under identical circumstances, on TSH-stimulated malic enzyme mRNA levels directly validate this hypothesis. We, thus, suggest that the opposite action of hydrocortisone on the two cAMP-mediated activities may reflect positive and negative regulation of the action of cAMP at different steps in the transduction process, one being transcriptional (DNA synthesis) and the other posttranscriptional (induction of iodide porter activity).

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1990

Epidermal growth factor stimulate cell proliferation and inhibits prolactin secretion of the human decidual cells in culture.

Epidermal growth factor (EGF) has been found to be mitogenic in a variety of tissues. We investigated the biological effect of EGF on early pregnant human decidua and the non-pregnant decidualized human endometrium in the primary cell culture. EGF had a stimulatory action on cell proliferation in the early pregnant decidual cells and an inhibitory effect on prolactin (PRL) secretion from the decidual cells. The addition of progesterone into culture medium suppressed cell proliferation of decidual cells, whereas it enhanced PRL secretion from decidua. The analysis of the specific receptor for EGF in the early pregnant decidua and non-pregnant decidualized endometrium revealed that both tissues had a single component EGF receptor with a dissociation constant of nM order. These results suggest that EGF may play a role in the growth and function of endometrial stromal cells.

Cell Division↗

PubMed1989

The arachidonic acid signal system in the thyroid: regulation by thyrotropin and insulin/IGF-I.

The present study and the previous report (6) show that the cyclooxygenase path is a primary route of metabolism of arachidonic acid in FRTL-5 rat thyroid cells. The production of PGD2 and PGE2 is an active process in intact cells treated with complete medium including TSH, insulin and 5% calf serum. In contrast, PGF2 alpha and HHT are probably nonenzymatic degradation products of an unstable intermediate, PGH2, since the two compounds are produced and occupy a significant proportion of the cyclooxygenase metabolites only in the homogenate system; this is true in other cells. Although the production of prostaglandins involves three steps, i.e. the release of free arachidonic acid, the production of PGH2 by PGH synthase (cyclooxygenase) and the conversion of PGH2 to various prostaglandins by specific isomerases or synthetases, the first step, the release of free arachidonic acid, has been, until recently, believed to be the sole step important for the regulation of prostaglandin synthesis. This presumption rested on the following observations. Only the free form of arachidonic acid is converted to prostaglandins and the intracellular free arachidonic acid pool is very small compared to the esterified form in phospholipids. The size of the free arachidonic acid pool is regulated by the balance between release from phospholipids by phospholipases and reacylation into phospholipids. When resting cells are stimulated, the release of arachidonic acid and the production of prostaglandins increase concomitantly. The present study shows, however, that all three steps of prostaglandin synthesis are under regulatory control in FRTL-5 rat thyroid cells and that the control is a complex process involving TSH, insulin/IGF-I, and serum. The first step is primarily under the control of TSH. TSH increases the synthesis of arachidonic acid and also, like norepinephrine (5, 6) induces the release of arachidonic acid from the cell by a mechanism involving a pertussis toxin-sensitive G protein. Regulation of the second step can be estimated by measuring cyclooxygenase activity. The present report shows that TSH increases cyclooxygenase activity, presumably by increasing gene expression, but that the TSH effect on cyclooxygenase activity requires insulin/IGF-I or serum. This result is similar to studies showing the effect of TSH and insulin/IGF-I on glycosaminoglycan synthesis, thyroglobulin synthesis, and growth in FRTL-5 thyroid cells.(ABSTRACT TRUNCATED AT 400 WORDS)

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↗

PubMed1988

Effects of transforming growth factor-beta on deoxyribonucleic acid synthesis and iodine metabolism in porcine thyroid cells in culture.

The effect of transforming growth factor (TGF)-beta on DNA synthesis and iodine metabolism was studied in cultured porcine thyroid cells. TGF-beta dose-dependently inhibited DNA synthesis stimulated by both insulin-like growth factor I and epidermal growth factor but did not affect the number or affinity of receptors for the two growth factors, suggesting that TGF-beta inhibits postreceptor events responsible for initiation of DNA synthesis. TGF-beta was a potent inhibitor of iodine metabolism. When porcine thyroid cells were cultured with TSH for 3 days in the presence of TGF-beta, TSH-induced iodide uptake and organification were reduced at rates that were dependent on the TGF-beta concentrations. The inhibition was detectable at TGF-beta concentrations as low as 50 pg/ml, and complete suppression was seen at 1 ng/ml. Only 6 h of exposure to TGF-beta resulted in a significant inhibition of TSH-induced iodine metabolism. Treatment of thyroid cells with TGF-beta for 3 days did not reduce cAMP production stimulated by TSH. Moreover, the intracellular cAMP level of thyroid cells cultured with TSH plus TGF-beta did not differ from that of cells cultured with TSH alone. TGF-beta decreased iodide uptake stimulated by forskolin or 8-bromo-cAMP. These results strongly suggest that TGF-beta inhibits TSH-stimulated iodine metabolism, at least in part, by affecting events subsequent to cAMP production. The physiological role of TGF-beta remains to be determined, but it may be involved in the regulation of thyroid cell growth and function.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1988

High serum progesterone in hyperthyroid men with Graves' disease.

We measured serum progesterone in five men with hyperthyroidism due to Graves' disease. All had elevated serum progesterone levels before treatment with an antithyroid drug, and their serum progesterone levels declined concomitantly with their serum thyroid levels during treatment. Progesterone enhances estrogen's stimulation of mammary gland growth, and our findings suggest that progesterone may play a role in the gynecomastia that occurs in men with hyperthyroidism.

Adult↗

PubMed1988

Thyroid volume and serum thyroglobulin levels in patients with acromegaly: correlation with plasma insulin-like growth factor I levels.

The pathogenesis of the goiter that is frequently found in patients with acromegaly is not known. Using ultrasonic scanning, we measured thyroid volume in 17 euthyroid patients with acromegaly and examined the relationships among thyroid size, plasma GH and insulin-like growth factor (IGF-I) levels, and serum thyroglobulin (TG) levels. The mean estimated thyroid volume in these 17 patients was 32.8 +/- 15.5 (+/- SD) mL, significantly larger than that in normal subjects (15.4 +/- 3.1 mL), and 64.7% of the patients had multinodular goiter, as identified by ultrasonography. Thyroid volume was positively correlated with plasma GH and IGF-I levels and heel-pad thickness, but not with the serum TSH level. In 7 patients, thyroid volume decreased in association with a decline in plasma GH and IGF-I levels after surgical treatment. The serum TG level was elevated in 7 of the 15 patients in whom it was measured, and the mean value was 51.7 +/- 62.7 (+/- SD) micrograms/L (normal, 12.6 +/- 6.4 micrograms/L). We found no correlations among the serum TG and TSH levels, plasma GH and IGF-I concentrations, and/or thyroid volume. However, serum TG decreased after surgical treatment, just as did plasma IGF-I. These observations together with the results of recent in vitro studies by others suggest that IGF-I is one of the factors involved in goiter formation, but the elevated serum TG levels in acromegaly are controlled not only by IGF-I but also by other factors.

Acromegaly↗

PubMed1988

The inhibitory effect of iodide on growth of rat thyroid (FRTL-5) cells.

The effect of iodide on growth of rat thyroid cells (FRTL-5) was studied. TSH-stimulated cell growth was inhibited by iodide in a concentration-dependent manner, and an effect of iodide was detected at 10(-6) mol/l. KClO4 or 1-methylimidazole-2-thiol blocked the effect of iodide, suggesting that iodide uptake and its organification are required to produce the inhibitory effect of iodide on cell growth. Iodide not only decreased TSH-stimulated cAMP production in FRTL-5 cells but also cell growth induced by cAMP. These observations suggest that iodide inhibits TSH-stimulated growth of the cells by attenuating cAMP production and also by acting on the step(s) distal to cAMP generation. The inhibitory effect of iodide was also seen in growth stimulated by insulin, insulin-like growth factor-I or 12-O-tetradecanoyl phorbol 13-acetate, suggesting multiple sites of action of iodide in the process of growth of FRTL-5 cells.

Animals↗

PubMed1987

Delayed transneuronal death of substantia nigra neurons prevented by gamma-aminobutyric acid agonist.

In an investigation of the mechanism by which brain lesions result in delayed degeneration of neurons remote from the site of injury, neurons within the caudate nucleus of rats were destroyed by local injection of the excitotoxin ibotenic acid. Treatment resulted in the rapid degeneration of the striatonigral pathway including projections containing the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) and delayed transneuronal death of neurons in the substantia nigra pars reticulata. The distribution of nigral cell loss corresponded to the loss of GABAergic terminals. Neuronal death was prevented by long-term intraventricular infusion of the GABA agonist muscimol. Delayed transneuronal degeneration may be produced by neuronal disinhibition consequent to loss of inhibitory inputs. Replacement of inhibitory transmitters by suitable drugs may prevent some forms of delayed neuronal death.

Animals↗

PubMed1987

Functional subdivisions of the nucleus tractus solitarii of the rat as determined by circulatory and respiratory responses to electrical stimulation of the nucleus.

We have recently identified two functional subdivisions of the nucleus tractus solitarii (NTS) in the cat: the pressor and apneustic (inspiratory) response zone, and the depressor and apneic (hypopneic) response zone. The presence of such functional subdivisions in the NTS of the rat was explored. The circulatory and respiratory responses to electrical stimulation of a small part of the NTS were surveyed in all regions of the NTS in rats anesthetized with urethane (500 mg/kg, i.p.). A set of depressor, bradycardiac and apneic responses was elicited in the dorsomedial portions, whereas a pressor response with or without an apneic (hypopneic) response was elicited in the ventral and lateral portions. The presence of a difference in the functional subdivisions between spontaneously hypertensive rats (SHR) and normotensive rats (WKY) was also explored. The SHR group showed significantly larger pressor responses than the WKY group (P less than 0.001), despite its higher basal level of arterial blood pressure, but significantly fewer bradycardiac responses (P less than 0.001). This suggests the SHR group is more sensitive in terms of sympathetic vasomotor activity but less sensitive in parasympathetic cardioinhibitory activity. There was no significant difference in the distribution of the pressor and depressor response zones between the two strains.

Animals↗

PubMed1987

Projections of supraspinal structures to the phrenic motor nucleus in rats studied by a horseradish peroxidase microinjection method.

Autonomic nervous functions are considered to be influenced by the system controlling respiratory function, and vice versa. Recently, it became apparent that there is an interaction between the nervous systems controlling circulatory and respiratory functions, but anatomical knowledge about these control systems is still scanty. To compensate this lack of anatomical knowledge about the location of respiratory neurons, supraspinal structures projecting to the phrenic motor nucleus were studied using rats for purpose of comparison with our previous findings in the cat. After the microinjection of a small amount of horseradish peroxidase (HRP) into the phrenic motor nucleus, which was physiologically identified, some 2151 HRP-labeled cells were identified in the 5 selected experiments. These cells were distributed in the medulla (88.7%) and pons (11.3%). The majority were concentrated in and around the two nuclei involved in the cardiovascular control system, i.e. the nucleus para-ambiguus (55.5%) which is known as a pressor-tachycardia-apnea response zone and nucleus retrofacialis (10.3%) which is known as a depressor-bradycardia-apnea response zone. This distribution was similar to that in the cat except that there were many labeled cells in the nucleus tractus solitarii of the cat (21.5%) but only a few in the rat (1.0%). The insignificant solitario-phrenic projection in the rat suggests a different organization of respiratory nuclei in this species.

Animals↗

PubMed1987

Interaction of insulin-like growth factor I with porcine thyroid cells cultured in monolayer.

The interaction of insulin-like growth factor I (IGF-I) with porcine thyroid cells cultured in monolayer was studied. Specific binding of [125I]iodo-IGF-I to thyroid cells was a reversible process dependent on the time and temperature of incubation. A steady state was achieved in 18 h at 4 C and averaged 14.2 +/- 2% (mean +/- SD)/10(6) cells. Binding of [125I]iodo-IGF-I was inhibited by unlabeled IGF-I; half-maximal inhibition occurred at concentrations of 2-5 ng/ml. Multiplication-stimulating activity (rat IGF-II) and pork insulin had relative potencies of 1:20 and 1:300 compared with IGF-I. Scatchard analysis of binding data revealed a single class of IGF-I receptors with a Ka of 4.3 X 10(10) M-1, 49,000 binding sites were estimated per cell. Affinity cross-linking and autoradiography demonstrated the presence of type I IGF receptors. Thyroid cells also had specific receptors for insulin, but specific binding of [125I]iodoinsulin (2.03 +/- 0.03%/10(6) cells) was much lower than that of [125I]iodo-IGF-I. Preincubation of thyroid cells with IGF-I or insulin caused a concentration-dependent decrease in [125I]iodo-IGF-I binding due to an apparent loss of receptors. Preincubation with epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, or TSH did not alter subsequent binding of [125I]iodo-IGF-I. Low concentrations of IGF-I stimulated DNA synthesis and proliferation of thyroid cells and acted synergistically with epidermal growth factor. Multiplication-stimulating activity and insulin had relative potencies in stimulating DNA synthesis comparable to their abilities to inhibit the binding of [125I]iodo-IGF-I to thyroid cells, suggesting that their effects are mediated primarily by IGF-I receptors. Preincubation with IGF-I did not alter cAMP responsiveness to TSH. We, thus, demonstrated the presence of functional and regulated IGF-I receptors on porcine thyroid cells.

Animals↗