Acetylation with succinimidyl acetate affects both the catalytic site and the regulation of the erythrocyte Ca2+ pump

Acetylation of lysine residues of the erythrocyte Ca2+ pump using succinimidyl acetate (SA) led to its complete inactivation. In the absence of any of the major activators of the pump (namely calmodulin and acidic phospholipids), ATP fully protected the pump from inactivation by SA, with a K0.5 of 13 microM. This value is very close to the Km of the high-affinity site for ATP, thus suggesting that the residue(s) involved is(are) near or at the catalytic site of the Ca(2+)-ATPase. Furthermore, the presence of 500 microM ATP prevented the acetylation of about two residues per molecule of enzyme. Acetylation by SA also prevented the activation of the Ca2+ pump by calmodulin, acidic phospholipids or controlled trypsin proteolysis. This effect of SA treatment was not avoided by the presence of ATP in the preincubation medium, indicating a second set of modified residues. The fact that the three modes of activation were cancelled in a similar fashion by SA suggests that, although acting via different mechanisms, they share at least a common step in which SA-sensitive lysine residues may participate. Moreover, modification of the pump by SA plus ATP decreased the KCa when the activity was measured in both the absence and presence of calmodulin, suggesting that the residue(s) modified in this case is(are) involved directly in the regulation of the affinity for Ca2+.

Insulin-like growth factor-I production and action in porcine thyroid follicular cells in monolayer: regulation by transforming growth factor-β

ABSTRACT The regulation of thyroid follicular cell growth in vitro involves autocrine or paracrine actions of insulin-like growth factor-I (IGF-I), which are partially suppressed by transforming growth factor-β (TGF-β). Using subconfluent monolayers of porcine thyroid follicular cells, the aims of this study were to establish whether the actions of TGF-β involve changes in the synthesis of, or response to, IGF-I. We also investigated the extent to which inhibitory actions of iodide on IGF-I-dependent proliferation of thyroid follicular cells may be attributable to the production of TGF-β by follicular cells, as opposed to iodide-mediated autoregulation events. Exposure of porcine thyroid follicular cells in subconfluent monolayer culture to TGF-β over a 7-day period reduced both IGF-I release and the incorporation of [met hyl-3H]thymidine into trichloroacetic acidprecipitable cellular material, while preincubation of cells with NaI (0 ·1 mmol/l) for 24 h prior to the addition of TSH reduced the stimulatory effect of the latter on IGF-I release over the following 7 days. Preincubation of cells with iodide also reduced basal (i.e. autonomous) [methyl-3H]thymidine incorporation. This effect was partially reversed when, following initial exposure to follicular cells, iodide-containing preincubation medium was immunoadsorbed with a neutralizing TGF-β antiserum, and subsequently readded to the cells. Furthermore, similar immunoadsorption of iodide-free preincubation medium resulted in an enhancement of the control level of [methyl-3H]thymidine incorporation when the treated medium was returned to the original cultures. The results of this study are consistent with the hypothesis that IGF-I and TGF-β are both produced by subconfluent thyroid follicular cells in vitro, and that the inhibitory action of TGF-β on follicular cell growth may involve a decrease in the thyroidal production of IGF-I. While the attenuating action of iodide on follicular cell proliferation may, in part, reflect an increased autocrine production of TGF-β, and a reduction by TGF-β of the growth response to IGF-I, these studies also provide evidence that the intrathyroidal actions of TGF-β include an attenuation of IGF-I biosynthesis. Journal of Endocrinology (1991) 130, 3–9

Effects of Ca2+ and Mg2+ on ATP-dependent 45Ca2+ influx in A-431 human epidermoidal carcinoma cells

Upon stimulation with 10−6–10−3 M ATP, A-431 human epidermoidal carcinoma cells incorporated radioactive calcium from their medium in a temperature-dependent manner. The rate of incorporation of 45Ca2+ was rapid for the initial 5 min, but decreased immediately thereafter. The preincubation of cells for 2 h in medium depleted of both Ca2+ and Mg2+ abolished the ATP-dependent 45Ca2+ incorporation, irrespective of whether or not the subsequent incubation medium contained Mg2+ ions. ATP-dependent 45Ca2+ incorporation could be restored by a second preincubation (1 h) in medium containing 1 mM Mg2+, but no Ca2+. The Mg2+ ions in the second preincubation medium could be replaced by Ca2+, Co2+, or Cu2+ for restoration of such activity. Elevation of inositol trisphosphate (InsP3) was observed in cells depleted of either Ca2+ or Mg2+, but not in cells depleted of both ions. A parallel effect was observed in changes in [Ca2+]i. Since the concentration of cytosolic calcium ions does not change by incubation of cells in medium depleted of and (or) restored with calcium ions, we conclude that either calcium or magnesium ions associated with some cellular component(s) are responsible for production of InsP3, which then supposedly mobilizes Ca2+ and provokes 45Ca2+ influx.Key words: intracellular calcium ion, inositol trisphosphate, calcium influx.

Effects of dopamine and morphine on immunoreactive somatostatin and LH-releasing hormone secretion from hypothalamic fragments in vitro

ABSTRACT Dopamine and morphine modulate GH and LH release, probably at a hypothalamic locus. To investigate this in more detail, we studied the influence of these substances on somatostatin and LH-releasing hormone (LHRH) release from rat hypothalamic fragments in vitro. Hypothalamic fragments were incubated in Earle's medium. After 60 min of preincubation, medium from two 20-min incubations was collected and somatostatin and LHRH levels measured by radioimmunoassay. Dopamine (10 nmol/l–0·1 mmol/l) induced a progressive increase (r = 0·41; P <0·01) in basal somatostatin levels. K + (30 mmol/l)-induced somatostatin release was also increased (r = 0·54; P <0·01) by increasing doses of dopamine. Metoclopramide (10 μmol/l) blocked the dopamine (1 μmol/l)-induced increase in somatostatin release. No significant relationship between dopamine and LHRH was found either basally or after K + (30 mmol/l) stimulation. Basal somatostatin was negatively correlated (r = −0·63; P <0·01) with morphine concentrations. No significant correlation was found after K+ (30 mmol/l) depolarization. Basal LHRH release was not influenced by morphine, while K +(30 mmol/l)-induced release was significantly lower than controls only at a concentration of 10 nmol/l. These results suggest that dopamine and morphine act at a hypothalamic level to modulate GH release through alterations in somatostatin secretion. Dopamine and morphine have no consistent effect on hypothalamic LHRH release. J. Endocr. (1985) 106, 317–322

Insulin stimulates granulosa cells: increased progesterone and cAMP production in vitro

Granulosa cells (GC) harvested from large (6-12 mm) follicles of cycling pigs obtained at the abattoir were incubated in the absence or presence of insulin and/or human chorionic gonadotropin (hCG). The total accumulation of progesterone and cAMP in the tissue plus medium was quantified by radioimmunoassay of hexane extracts and trichloroacetic acid extracts, respectively, and was related to cellular DNA content. Insulin (1 microgram/ml) had little effect on progesterone accumulation by freshly harvested GC incubated for up to 26 h at 37 degrees C. After preincubation of GC for 16 h in the absence of hormones, insulin (650 ng/ml) only enhanced subsequent basal progesterone accumulation after an additional 22 h. However, preincubation of cells for 20 h in the presence of 1 microgram/ml of insulin increased subsequent progesterone accumulation in both the absence and the presence of hCG (50 ng/ml). The effect of a 20-h preincubation with insulin on subsequent progesterone accumulation was related to the insulin concentration. At a maximally effective concentration of 5 micrograms/ml during the preincubation period, insulin increased subsequent basal and hCG-stimulated progesterone accumulations more than twofold. The concentrations of insulin in the preincubation medium that produced half maximal stimulation (ED50) were 300 and 200 ng/ml for subsequent basal and hCG-stimulated progesterone accumulations, respectively. After correction for loss of insulin immunoreactivity in the incubation medium, the ED50 levels were 60 and 50 ng/ml, respectively. Insulin elevated hCG-stimulated progesterone accumulation over the entire range of hCG concentrations; the effect on hCG-stimulated cAMP production was evident only at higher hCG concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

MgATP-induced inhibition of the adenosine triphosphatase activity of the chloroform-released mitochondrial adenosine triphosphatase

1. Preincubation of the ox heart chloroform-released mitochondrial ATPase with MgATP results in a time-dependent inhibition of ATPase activity. No re-activation occurs when MgATP remains in the preincubation medium. The enzyme activity returns when all the MgATP in the preincubation system has been hydrolysed. 2. The mechanism of the MgATP-induced inhibition was examined. Inhibition occurs on incubation with MgATP or other hydrolysable nucleotides. Incubation with MgADP or Pi does not cause any inhibition. Neither freshly bound adenine nucleotide nor Pi is associated with inhibited enzyme. The rate of MgATP-induced inhibition correlates with the rate of ATP hydrolysis in the preincubation medium. Changing the rate of ATP hydrolysis at a fixed concentration of ATP also changes the rate of MgATP-induced inhibition by the same proportion. The inhibition is thus related to the ATP-hydrolysis process itself. 3. We propose that intermediate enzyme species of the ATP-hydrolytic sequence can undergo a conformational change to form inhibited species. The kinetics of the inhibition suggest that a substrate-activation step is involved in ATP hydrolysis and MgATP-induced inhibition. 4. The effects of the nature of the preincubation medium on the process of MgATP-induced inhibition and its reversal were examined.

Modulation of Amino Acid Transport in Rat Liver Slices by Tissue Preincubation

In rat liver slices, a prolonged preincubation resulted in a stimulation of AIB and alanine uptake. The increase of transport of both amino acids can be ascribed to a reduction of Kt without an alteration of Vt. The enhancement of transport seems to be restricted to the A mediation as indicated by the findings that only the Na+-dependent, MeAIB-inhibitable portion of AIB transport was increased by the prolonged preincubation, and, furthermore, that the uptake of cysteine, a specific substrate for system ASC in liver cells, was not affected. The effect of an extended preincubation on AIB transport was abolished by the addition of high concentrations of AIB and alanine to the preincubation medium. Several other amino acids tested did not affect the increase of AIB transport. The observed stimulation of transport seems not to be due to the derepression of the synthesis of transport proteins, as cycloheximide and 5-azacytidine did not block the increase of AIB uptake. The enhancement of amino acid transport does not appear to be a result of a release from transinhibition since the uptake of alanine was markedly enhanced after 3 h of preincubation while the alanine concentrations in the incubated liver slices did not change significantly.

T-Lymphocyte precursors. I. Synergy between precursor and mature T lymphocytes in the response to concanavalin A.

When mouse bone marrow cells are mixed with cortisol-resistant thymocytes and stimulated in vitro with concanavalin A, the mitogenic response observed is much greater than additive, that is, it is synergistic. Between 94 and 96% of responding cells could be identified as T cells (Thy-1 positive) and of these, 79-100% derived from the cortisol-resistant thymocyte population, not from the bone marrow. Purified macrophages could not replace bone marrow; and marrow depleted of mature T or B cells worked as well as normal marrow. Thus, T and B cells and macrophages were ruled out as the synergizing cell of bone marrow. Nude spleen contained 10 times as many precursors of T cells as did nude marrow and was 10 times better at synergy with cortisol-resistant thymocytes. This implication of the pre-T cell as synergizer was supported by the finding that the synergistic activity of marrow was lost on preincubation, but maintained if the preincubation medium contained thymosin or cyclic AMP. Thus, the ability to enhance the response of relatively mature T cells to Con A is a property of pre-T cells. It is anticipated that this property will allow more detailed studies of T-cell precursor development in mice, and possibly in man.