Epidermal growth factor (EGF) produces inositol phosphates and increases cytoplasmic free calcium in cultured porcine thyroid cells

The initial signal for thyroid cell proliferation is unknown. This is the first report to show that epidermal growth factor (EGF) produces inositol phosphates and increases cytoplasmic free calcium ([Ca2+]i) in the thyroid gland. In cultured porcine thyroid cells, 10 nM EGF produces a breakdown of phosphatidylinositol and stimulates inositol phosphate production. Ten nM EGF increases [Ca2+]i, measured using fura-2, a fluorescent Ca2+ indicator; the EGF-induced [Ca2+]i response occurs immediately, reaches a maximum within several seconds, and then slowly declines. EGF stimulates production of inositol phosphates, which seem to increase [Ca2+]i. Inositol phosphate production and an increase in [Ca2+]i after EGF-stimulation may function as an initial signal for thyroid cell proliferation.     

Insulin-like growth factor I stimulates inositol phosphate accumulation, a rise in cytoplasmic free calcium, and proliferation in cultured porcine thyroid cells

Insulin-like growth factor (IGF-I) stimulates thyroid cell proliferation. Using primary cultured porcine thyroid cells, we studied the intracellular pathways that mediate the action of IGF-I on thyroid cell proliferation. IGF-I stimulates inositol phosphate accumulation, a rise in cytoplasmic free calcium [( Ca2+]i), and cell proliferation. Exposure to IGF-I results in a time- and dose-dependent accumulation of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate. IGF-I also increases [Ca2+]i, measured using fura-2, a fluorescent Ca2+ indicator; the IGF-I-induced [Ca2+]i response occurs immediately, reaches a maximum within 1 min, and then slowly declines. IGF-I stimulates thyroid cell proliferation, stimulates thymidine incorporation, and increases cell numbers. The IGF-I-induced inositol phosphate accumulation and [Ca2+]i response parallel thyroid cell proliferation in a dose-dependent manner; the maximal response is observed at a concentration of 100 ng/ml IGF-I, with half-maximal stimulation at approximately 10 ng/ml. Inositol phosphate accumulation and [Ca2+]i response after IGF-I stimulation may function as intracellular messengers for thyroid cell proliferation. This report may constitute the first demonstration of IGF-I-stimulated inositol phosphate accumulation and [Ca2+]i response in the cells.     

Noninactivation of thyrotropin by cultured porcine thyroid cells.

Freshly isolated porcine thyroid cells were cultured in the presence of highly purified porcine thyrotropin. Cells associate into follicles between the second and tenth day of culture and later form a monolayer. The biological and immunological activity of thyrotropin was measured daily in the media. Thyrotropin concentration and biological activity remained unchanged from the onset of the culture up to day 14. Limiting factors influencing thyroglobulin biosynthesis do not appear before day 13. The loss of follicular organization at day 10 cannot be explained by thyrotropin degradation in the medium. Considering the number of receptors per cell and the half life of the thyrotropin . receptor complex in the two dissociation compartments previously demonstrated, it appears in terms of both biological activity and affinity for the receptors that the thyrotropin molecules released from the first compartment do not differ from native molecules. It can be calculated that at least 31% of the molecules released from the second compartment are not inactivated. Thus, it is probable that the catabolism of thyrotropin on the receptor, or near the receptor site, does not play an important role in the regulation of thyroid cell function in vitro.     

Mechanism of H2O2 production in porcine thyroid cells: evidence for intermediary formation of superoxide anion by NADPH-dependent H2O2-generating machinery.

Hydrogen peroxide (H2O2), which is required for thyroid hormone synthesis, has been believed to be produced at the apical cell surface of thyroid follicular cells. However, we recently found that plasma membrane from porcine thyroid exclusively generated superoxide anion (O2-) by employing a novel method for simultaneous determination of H2O2 and O2- with diacetyldeuterioheme-substituted horseradish peroxidase (diacetyl-HRP) as the trapping reagent [Nakamura, Y., Ohtaki, S., Makino, R., Tanaka, T., & Ishimura, Y. (1989) J. Biol. Chem. 264, 4759-4761]. The present study describes the mechanism of H2O2 production as analyzed by this new method. Incubation of cultured porcine follicular cells with ionomycin, a Ca-ionophore, caused an increase in oxygen uptake of about 80%. During enhanced respiration, the cells released H2O2 in an amount equivalent to the amount of oxygen consumed as judged by the formation of compound II of diacetyl-HRP, and H2O2 adduct of the peroxidase. No formation of compound III of the peroxidase, an O2- adduct, was detected during burst respiration. Thus, the intact cells exclusively released H2O2 to the outside of the cells. On the other hand, when the cell fragments from follicular cells were incubated with NADPH or NADH in the presence of Ca2+, the production of O2- was observed only during NADPH-dependent burst respiration, supporting our previous results that the plasma membrane exhibited NADPH-dependent O2(-)-generating activity. O2- production by the plasma membrane was further confirmed by analyses of the effects of superoxide dismutase (SOD) and catalase on the reaction. These results suggested that H2O2 is secondarily produced through the dismutation of O2-.(ABSTRACT TRUNCATED AT 250 WORDS)     

H2O2 generation is decreased by calcium in isolated brain mitochondria

Release of H(2)O(2) in response to Ca(2+) loads (1-100 microM) was investigated using Amplex red fluorescent assay in isolated guinea-pig brain mitochondria respiring on glutamate plus malate or succinate. In mitochondria challenged with Ca(2+) (10 microM), in the absence of adenine nucleotides and inhibitors of the respiratory chain, the rate of H(2)O(2) release, taken as an indication of H(2)O(2) production, was decreased by 21.8+/-1.6% in the presence of NADH-linked substrates and by 86.5+/-1.8% with succinate. Parallel with this, a Ca(2+)-induced loss in NAD(P)H fluorescence, sustained depolarization, decrease in fluorescent light scattering signal and in calcein fluorescence were detected indicating an increased permeability and swelling of mitochondria, which were prevented by ADP (2 mM). In the presence of ADP H(2)O(2) release from mitochondria was decreased, but Ca(2+) no longer influenced the generation of H(2)O(2). We suggest that the decreased H(2)O(2) generation induced by Ca(2+) is related to depolarization and NAD(P)H loss resulting from a non-specific permeability increase of the mitochondrial inner membrane.     

APP processing is regulated by cytoplasmic phosphorylation

Amyloid-beta peptide (Abeta) aggregate in senile plaque is a key characteristic of Alzheimer's disease (AD). Here, we show that phosphorylation of amyloid precursor protein (APP) on threonine 668 (P-APP) may play a role in APP metabolism. In AD brains, P-APP accumulates in large vesicular structures in afflicted hippocampal pyramidal neurons that costain with antibodies against endosome markers and the beta-secretase, BACE1. Western blot analysis reveals increased levels of T668-phosphorylated APP COOH-terminal fragments in hippocampal lysates from many AD but not control subjects. Importantly, P-APP cofractionates with endosome markers and BACE1 in an iodixanol gradient and displays extensive colocalization with BACE1 in rat primary cortical neurons. Furthermore, APP COOH-terminal fragments generated by BACE1 are preferentially phosphorylated on T668 verses those produced by alpha-secretase. The production of Abeta is significantly reduced when phosphorylation of T668 is either abolished by mutation or inhibited by T668 kinase inhibitors. Together, these results suggest that T668 phosphorylation may facilitate the BACE1 cleavage of APP to increase Abeta generation.     

BAY-K-8644, a calcium channel agonist, induces a rise in cytoplasmic free calcium and iodide discharge in thyroid cells

BAY-K-8644, a calcium channel agonist, induces a rise in cytoplasmic free calcium and iodide discharge in cultured porcine thyroid cells. The cytoplasmic free calcium concentration, [Ca2+]i, was measured using aequorin, a calcium-sensitive photoprotein. BAY-K-8644, a dihydropyridine derivative, acts as a Ca channel agonist and induces a rise in [Ca2+]i and iodide discharge; 0.5 nM BAY-K-8644 is a minimal dose to effect a rise in [Ca2+]i and iodide discharge and 50 nM BAY-K-8644 produces the maximal effect. The data indicate that BAY-K-8644-induced iodide discharge is mediated by a rise in [Ca2+]i.     

Cytoplasmic pH in cultured porcine thyroid cells: alkalinization by epidermal growth factor

We studied the effects of epidermal growth factor (EGF), thyroid-stimulating hormone (TSH) and amiloride on cytoplasmic pH (pHi) in cultured porcine thyroid cells. We used 2',7'-bis(2-carboxyethyl)-5- (and 6-)carboxyfluorescein (BCECF), an internalized fluorescent pH indicator, to measure pHi. EGF stimulated thyroid cell alkalinization and proliferation, which were blocked by amiloride. EGF-stimulated thyroid cell alkalinization depended on extracellular Na+ concentrations. EGF stimulation resulted in an activation of Na+/H+ exchange, which alkalinized the cells. The results indicated that Na+/H+ exchange or cell alkalinization might function as a transmembrane signal transducer in the action of EGF. In the present system, TSH did not stimulate alkalinization or proliferation.     

Lithium alters elicitor-induced H2O2 production in cultured plant cells

Lithium pollution may seriously influence the metabolic and signalling processes of plants. In the present paper, we investigate the effect of lithium chloride on fungal elicitor-triggered H₂O₂ generation in Rubia tinctorum L. cell cultures. Our results show that Li⁺ strongly influences elicitor-induced H₂O₂ formation and time-course in the cells nad culture medium. Neomycin, a phospholipase C inhibitor, and 2-APB, an inositol-1,4,5-triphosphate (IP₃) receptormediated Ca²⁺ release blocker, strongly affected the elicitor-induced H₂O₂ production and had a similar effect on elicitor-triggered H₂O₂ formation as Li⁺. We monitored changes in H₂O₂ location at subcellular level and our observations confirmed the changes measured by quantitative methods. The obtained results enabled us to deduce that the IP₃ pathway might be involved in the early signalling events leading to the moderation of elicitor-induced reactive oxygen species generation.     

Generation of oxygen free radicals in thyroid cells and inhibition of thyroid peroxidase

We examined whether superoxide (O(2)(-)) is produced as a precursor of hydrogen peroxide (H(2)O(2)) in cultured thyroid cells using the cytochrome c method and the electron paramagnetic resonance (EPR) method. No O(2)(-) or its related radicals was detected in thyroid cells under the physiological condition. The presence of quinone, 2,3-dimethoxy-l-naphthoquinone (DMNQ), or 2-methyl-1, 4-naphthoquinone (menadione), in the medium produced O(2)(-) and hydroxyl radicals (OH*); the amount of H(2)O(2) generation was also increased. Incubation of follicles with DMNQ or menadione inhibited iodine organification (a step of thyroid hormone formation) and its catalytic enzyme, thyroid peroxidase (TPO). This inhibition should be caused by reactive oxygen species because the two quinones, particularly DMNQ, exert their effect through the generation of reactive oxygen species. It is speculated that the site-specific inactivation of TPO might have occurred at the heme-linked histidine residue of the TPO molecule, a critical amino acid for enzyme activity because OH* (vicious free radicals) can be formed at the iron-linked amino acid. TPO mRNA level and electrophoretic mobility of TPO were not inhibited by quinones. Our study suggests that thyroid H(2)O(2) is produced by divalent reduction of oxygen without O(2)(-) generation. If thyroid cells happen to be exposed to significant amount of reactive oxygen species, TPO and subsequent thyroid hormone formation are inhibited.     

Elevation of cytosolic free calcium by platelet-activating factor in cultured rat mesangial cells

Rat glomerular mesangial cell monolayers loaded with the fluorescent probe fura-2 responded to exogenous platelet-activating factor (PAF) with a rapid increase in cytosolic free calcium concentration ([Ca2+]i). PAF-induced [CA2+]i transients consisted of a dose-dependent phasic peak response followed by a sustained tonic phase of increased [Ca2+]i. Chelation of extracellular calcium with EGTA suppressed the tonic phase of increased [Ca2+]i but did not affect the phasic peak response. This suggests two mechanisms for the elevation of [Ca2+]i: a transient mobilization from intracellular stores and an enhanced calcium influx across the plasma membrane, possibly mediated by receptor-operated channels. Lyso-PAF had no effect on basal [Ca2+]i and the PAF-receptor antagonist L652,731 selectively inhibited responses to PAF. PAF-stimulated mesangial cells displayed homologous desensitization to reexposure to PAF while still being responsive to other calcium-mobilizing agonists. Preincubation of cells with the protein kinase C (PKC) activator phorbol myristate acetate diminished the PAF-induced [Ca2+]i transient, suggesting a regulatory role for PKC in PAF-activation of mesangial cells. An increase in [Ca2+]i, as a result of receptor-linked activation of phospholipase C, may mediate PAF-induced hemodynamic and inflammatory events in renal glomeruli.     

Albumin-bound lipids induce free cytoplasmic calcium oscillations in human osteoblast-like cells

[Ca(2+)](i) oscillations were found in human osteoblast-like cells (hOB cells) exposed to high-lipid bovine serum albumin (BSA), but not when exposed to low-lipid BSA. These [Ca(2+)](i) oscillations were inhibited by heptanol and suramin, which implies that gap junctions and purinergic signalling may be important for these [Ca(2+)](i) oscillations. The high-lipid BSA preparation that was used contains arachidonic acid. [Ca(2+)](i) oscillations could be induced by low lipid albumin with arachidonic acid added. The albumin-bound lipids were also important for osteoblast growth since DNA synthesis and the total cell protein content was higher in hOB cells exposed to high-lipid BSA. The effect of arachidonic acid on hOB cell proliferation was bone-donor dependent; both stimulatory and inhibitory effects were observed. The physiological importance of albumin-bound lipids is unclear; given that albumin has only minimal contact with osteoblasts under normal conditions. Only when bone capillaries are disrupted, e.g. during a fracture, would significant amounts of albumin reach osteoblasts. Albumin-bound lipids could therefore contribute to stimulation of osteoblast proliferation during fracture healing.     

Na(+)-ionophore, monensin-induced rise in cytoplasmic free calcium depends on the presence of extracellular calcium in FRTL-5 rat thyroid cells

Calcium is an important regulator of cell function, and may be influenced by the intracellular sodium content. In the present study, the Na(+)-ionophore, monensin, was used to investigate the interrelationship between changes in intracellular Na+ concentration ([Na+]i) and elevation of cytosolic Ca2+ concentration ([Ca2+]i) in FRTL-5 thyroid cells. Cytoplasmic Ca2+ levels were measured using the fluorescent dye, indo-1. Monensin induced a dose-dependent increase in [Ca2+]i in FRTL-5 cells. Inhibitors of intracellular Ca2+ release, TMB-8 and ryanodine, were unable to prevent the monensin effect on [Ca2+]i. The alpha 1-receptor antagonist, prazosin, did not block the monensin-stimulated increase in [Ca2+]i. In the absence of extracellular calcium there was a marked diminution in the monensin effect on [Ca2+]i, yet calcium channel antagonists (nifedipine, diltiazem and verapamil) did not inhibit the response. Replacement of Na+ by choline chloride in the medium depressed the monensin-evoked rise in [Ca2+]i by up to 84%. Furthermore, addition of the Na(+)-channel agonist, veratridine, elicited an increase in [Ca2+]i, even though less dramatic than that caused by monensin. Ouabain increased the resting cytosolic Ca2+ concentration as well as the magnitude of the monensin effect on [Ca2+]i. The absence of any effect on the Na(+)-ionophore evoked increase in [Ca2+]i upon addition of tetrodotoxin (TTX) excluded a possible involvement of TTX-sensitive Na+ channels. These data show that the rise in [Ca2+]i induced by increasing [Na+]i is largely dependent on both external Na+ and Ca2+. Calcium entry appears not to involve voltage-dependent or alpha 1-receptor sensitive Ca2+ channels, but may result from activation of an Na(+)-Ca2+ exchange system.     

Alpha 1-adrenergic stimulation and cytoplasmic free calcium concentration in cultured renal proximal tubular cells: evidence for compartmentalization of quin-2 and fura-2

This study was designed to examine the role of changes in cytoplasmic free calcium concentration ([Ca2+]i) during the response to alpha 1-adrenergic agonists in cultured renal proximal tubular cells. Experiments were carried out on primary cultures of canine proximal tubular cells grown in defined culture medium on a solid support, on collagen-coated polycarbonate membranes, or on collagen-coated glass coverslips. Quin-2 and fura-2 were used to monitor [Ca2+]i. The basal level of [Ca2+]i was 101 nM, as measured with quin-2, and 122 nM, as determined using fura-2. Fluorescence flow cytometry revealed that about 85% of the population of proximal tubular cells responded to phenylephrine with an increase in [Ca2+]i. Phenylephrine (10(-5) M) caused an immediate actual increase in [Ca2+]i by 18 and 24%, as determined with quin-2 and fura-2, respectively, with the peak increase in [Ca2+]i averaging 22% and 44% over the basal level (180-300 sec). This effect did not require extracellular calcium. The effect of phenylephrine was abolished by prazosin and verapamil. Fluorescence microscopy of quin-2 or fura-2 loaded cells revealed punctate areas of fluorescence within the cytoplasm suggesting vesicular uptake of the dyes. Pinocytotic entrapment of the dyes was demonstrated by the transfer of cell-impermeant fura-2 across tubular cell monolayers mounted in Ussing chambers. The transfer of the dye was similar to that of a marker of fluid-phase pinocytosis, Lucifer Yellow (LY). This pinocytotic entrapment of Ca2+-indicators would lead to underestimation of the actual calcium transients. Microfluorometric study of single proximal tubular cells "scrape-loaded" with fura-2 revealed a four-fold increase in [Ca2+]i concentration following stimulation with phenylephrine.     

Regulation of cytoplasmic free calcium by plant cell membranes

The free calcium concentration in the cytoplasm of higher plant cells is believed to be about 10⁻⁷M. The role of various membrane-borne calcium transporters present in plant tissues, together with chloroplasts and mitochondria, in maintaining this calcium concentration is reviewed, together with the role of various organelles in providing transient calcium fluxes upon stimulation.