Biomedical subjects

A Hagino

Publications and source records attributed to A Hagino.

8 recordsLinked to original sources

PubMed2001

Effects of adenosine 5'-triphosphate and growth hormone on cellular H+ transport and calcium ion concentrations in cloned bovine mammary epithelial cells.

The present experiment was carried out to investigate the effects of exogenous adenosine 5'-triphosphate (ATP) and growth hormone (GH) on cellular H(+) efflux rate (extracellular acidification rate) and Ca(2+) concentration ([Ca(2+)](c)) in cloned bovine mammary epithelial cells (bMEC) raised from the mammary gland of a 26-day-pregnant Holstein heifer. Perifusion of 2-day cultured cells with a medium containing ATP (10, 100 and 1000 micromol/l) for 30 min caused a significant and concentration-dependent increase in the cellular H(+) efflux rate. ATP application (100 micromol/l) caused a transient and large increase in [Ca(2+)](c) in all cells. In contrast, perifusion with a medium containing bovine GH at 10, 50 and 250 ng/ml for 30 min caused a significant decrease in the cellular H(+) efflux rate in a concentration-dependent manner. However, bovine GH application (50 ng/ml) caused a small decrease followed by an increase, in some cases, in [Ca(2+)](c). In bMEC treated with lactogenic hormones (1 microgram/l prolactin, 1 nmol/ml dexamethasone and 5 microgram/ml insulin) for 2 days, the increased H(+) efflux rate induced by ATP was significantly reduced, whereas the negative response induced by GH was inversely and significantly changed to the positive. Treatment of the cells with lactogenic hormones reduced the increase in [Ca(2+)](c) induced by ATP stimulation, while it enhanced the increase in [Ca(2+)](c) induced by GH stimulation. Application of ATP or GH did not cause any significant changes in [pH](c). Treatment with lactogenic hormones enhanced GH receptor (GHR) transcription that was determined by RT-PCR. From these results, we conclude that exogenous application of ATP and GH causes prompt and significant responses in H(+) transport and [Ca(2+)](c) that were significantly changed in the opposite direction by the treatment with lactogenic hormones. The lactogenic hormone treatment also enhanced GHR transcription, which may change post-receptor signal transduction systems for both agents in the bMEC.

Adenosine Triphosphate↗

PubMed1990

Nerve growth factor-induced transient increase in the phosphorylation of ribosomal protein S6 mediated through a mechanism independent of cyclic AMP-dependent protein kinase and protein kinase C.

Treatment of PC12h cells with nerve growth factor (NGF) induced a transient increase in the phosphorylation of a 35,000-dalton protein. This transient increase was observed also when extracts of NGF-treated cells were incubated with [gamma-32P]ATP. In the intact-cell phosphorylation system, treatment with N,2'-dibutyryladenosine 3',5'-cyclic monophosphate (dBcAMP) or 12-O-tetradecanoylphorbol 13-acetate (TPA) also induced a transient increase in the phosphorylation of the 35,000-dalton protein, but the effect was less than that of NGF. An effect comparable to that of NGF was obtained by the combination of dBcAMP and TPA. Pretreatment of PC12h cells with dBcAMP plus TPA for 3 days, which deprived the cells of their ability to respond to a rechallenge with dBcAMP, TPA, or dBcAMP plus TPA by increasing the rate of 35,000-dalton protein phosphorylation, caused only a slight attenuation of the NGF effect, directly indicating a minimal role of cyclic AMP (cAMP)-dependent protein kinase and protein kinase C in the mechanism of the NGF action. Pretreatment of the cells with K-252a, a protein kinase inhibitor, at a concentration of 300 nM almost completely blocked the action of NGF, but scarcely affected the action of dBcAMP, TPA, or dBcAMP plus TPA in intact-cell phosphorylation experiments. This NGF-sensitive 35,000-dalton protein was a ribosomal protein and identified as ribosomal protein S6. The results lead us to conclude that NGF activates some NGF-sensitive component(s), probably some specific protein kinase(s) other than cAMP-dependent protein kinase or protein kinase C, which is suppressed by K-252a and directly or indirectly activates a 35,000-dalton protein kinase(s) [S6 kinase(s)] to increase the rate of phosphorylation of the 35,000-dalton ribosomal protein (S6).

Adenosine Triphosphate↗

PubMed1990

Fibroblast growth factor-induced decrease in the phosphorylation of Nsp100 mediated through a calcium-dependent mechanism and blocked by lectins.

Separate treatment of PC12h cells with basic fibroblast growth factor (bFGF) and with epidermal growth factor (EGF) induced a selective decrease in the incorporation of radioactive phosphate into a 100,000-dalton soluble protein during phosphorylation with (gamma-32P)ATP of soluble extracts from the cells, as was seen previously with nerve growth factor (NGF). This 100,000-dalton soluble protein was designated in earlier studies as nerve growth factor-sensitive protein 100 (Nsp100). The inhibitory effects of bFGF and EGF on Nsp100 phosphorylation were prevented by pretreatment of PC12h cells with the calcium chelator, EGTA. Treatment of PC12h cells with the plant lectin wheat germ agglutinin (WGA), which binds to N-acetylglucosamine and sialic acid residues on glycoconjugates, blocked the inhibitory effects of bFGF, EGF, and NGF on Nsp100 phosphorylation. The blockage by WGA was reversed by the addition of the lectin-specific sugar N-acetylglucosamine to the PC12h cultures. Although pretreatment of PC12h cells with succinylated WGA, which has the ability to bind to N-acetylglucosamine but not to sialic acid residues, failed to block the inhibitory effect of NGF on Nsp100 phosphorylation as described previously, it did prevent the inhibitory effect of bFGF on this phosphorylation. These data suggest that in PC12h cells bFGF and EGF induce a decrease in the phosphorylation of Nsp100 mediated through a Ca2(+)-dependent mechanism, as in the case of NGF. Furthermore, the blockage of the bFGF-induced inhibition of Nsp100 phosphorylation by WGA and its succinylated form indicates that N-acetylglucosamine residues of bFGF receptor molecules might be involved in the mechanism by which bFGF inhibits the phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗

PubMed1989

Staurosporine-induced neurite outgrowth in PC12h cells.

Treatment of PC12h cells with staurosporine (100 nM), a potent inhibitor of protein kinases, promoted rapid outgrowth of neurites. The mechanism of neurite formation elicited by staurosporine is different from that elicited by nerve growth factor or by dibutyryl cyclic AMP, based on the independence from transcription or from activation of cyclic AMP-dependent protein kinase, respectively. Comparative experiments showed that of these three neurite-promoting agents, staurosporine was the most effective in eliciting neurite initiation.

Alkaloids↗

PubMed1989

Blockage of nerve growth factor action in PC12h cells by staurosporine, a potent protein kinase inhibitor.

Staurosporine, which has a structure similar to that of K-252a, a potent protein kinase inhibitor that blocks nerve growth factor (NGF) action in PC12 and PC12h cells, is also known as a potent inhibitor of several protein kinases. This study shows that in PC12h cells staurosporine has a dual action: at lower concentrations than that required by K-252a, it is an inhibitor of NGF induction of neurite formation and of changes in the phosphorylation of specific proteins, whereas at concentrations higher than that required to inhibit NGF-induced neurite outgrowth, it rapidly enhances outgrowth by itself.

Adrenal Gland Neoplasms↗

PubMed1989

Wheat germ agglutinin, concanavalin A, and lens culinalis agglutinin block the inhibitory effect of nerve growth factor on cell-free phosphorylation of Nsp100 in PC12h cells.

It has been shown that in PC12 and its subclone PC12h treatment of the cells with nerve growth factor (NGF) induces a selective decrease in the incorporation of radioactive phosphate into a 100,000-dalton protein, designated in an earlier study as Nsp100, in the subsequent phosphorylation of soluble extracts from cells with (gamma-32P)ATP. In the present study, we show that plant lectins, wheat germ agglutinin (WGA), concanavalin A (Con A), and lens culinaris agglutinin (LCA), inhibit the action of NGF on Nsp100 phosphorylation in PC12h cells. Treatment of the cells with WGA, which binds to N-acetylglucosamine and sialic acid residues on glycoproteins, strongly blocked the inhibitory action of NGF on the protein phosphorylation. Con A and LCA, both of which recognize the same specific sugars (mannose, glucose), displayed only a moderate blocking effect. Unlike the native lectin, succinylated WGA, which has the ability to bind to N-acetylglucosamine but not to sialic acid residues, and other lectins examined in this study did not inhibit the action of NGF on Nsp100. WGA-mediated inhibition of NGF action was reversed by the addition of N-acetylglucosamine and by the addition of a much lower concentration of a sialoglycoprotein, mucin, into the culture. Since the binding of succinylated WGA to N-acetylglucosamine residues of cell-surface glycoconjugates is not sufficient to prevent the action of NGF, WGA might act on sialic acid residues of the NGF receptor molecule to effect the inhibition of biological actions of NGF.

Adrenal Gland Neoplasms↗

PubMed1988

Adrenergic modulation of pancreatic glucagon and insulin secretion in sheep.

The effect of intravenous infusion of epinephrine, either alone or together with various doses of phentolamine or propranolol, on the secretion of both glucagon and insulin was determined in six sheep. Intravenous infusion of epinephrine alone caused increases in plasma glucagon and glucose concentrations and produced a slight but significant decrease in plasma insulin concentration. The concomitant infusion of propranolol and epinephrine augmented glucagon release and inhibited insulin secretion. Combined propranolol plus epinephrine infusion also caused a marked hyperglycemia. The concomitant infusion of phentolamine and epinephrine produced slight inhibition of glucagon secretion and markedly promoted insulin secretion. Epinephrine-induced hyperglycemia was eliminated by phentolamine infusion. The effects of isoproterenol infusion on plasma glucagon, insulin, and glucose concentrations were similar to that caused by the concomitant infusion of phentolamine and epinephrine. The effects of isoproterenol were abolished by the infusion of propranolol. It is concluded that an alpha-receptor mechanism is the most important component of adrenergic modulation of pancreatic glucagon secretion, whereas beta-receptor activation stimulates and alpha-receptor activation inhibits insulin secretion in sheep.

Adrenergic alpha-Antagonists↗

PubMed1984

Insulin response to glucose and glucose tolerance following feeding in sheep.

Sheep offered a roughage diet for 4 h daily were injected intravenously with glucose before and at various times after feeding. The insulin secretory response to glucose and the rate of disappearance of injected glucose were determined. While the basal concentration of plasma insulin was unchanged, the base-line plasma glucose concentration tended to decrease during the meal. The glucose load brought about an increase in the plasma insulin concentration at each injection, but the insulin response to glucose and the rate of glucose disposal were increased during the meal. On varying the time of feeding between 08.00 and 16.00 hours, the increase in the insulin response to glucose and in the rate of glucose disposal always appeared to be related to the giving of food, independent of the time food was offered. It is concluded that feeding increases the insulin response to an intravenous glucose load even when the increase in the basal level of plasma insulin on feeding is very modest in sheep given a roughage diet. The increased insulin response and glucose disposal rate following feeding did not appear to be related to diurnal rhythms in insulin secretory activity or glucose metabolism.

Animals↗