epidermal-growth-factor and Hypocalcemia

Extreme variations in extracellular Ca2+ concentrations ([Ca2+]e) modify the signaling generated by many hormones and growth factors. However, the influence of physiological changes in [Ca2+]e on the response to hepatic mitogens remains largely unknown. To study the influence of [Ca2+]e on the response to epidermal growth factor (EGF), hepatocytes from normal rat livers were equilibrated in vitro at [Ca2+]e similar to those observed in normocalcemia or hypocalcemia. To further investigate the effect of hypocalcemia in vivo, hepatocytes were obtained from chronically hypocalcemic rats and kept in vitro at the [Ca2+]e prevailing in vivo. Intracellular Ca2+ concentrations ([Ca2+]i) and DNA synthesis were evaluated after increasing doses of EGF. [Ca2+]e strongly influenced the [Ca2+]i response to EGF with significantly smaller [Ca2+]i increases in hepatocytes of normal rats kept in low [Ca2+]e compared with those kept in normal [Ca2+]e. In hypocalcemic rat hepatocytes, the response was further decreased and found to be significantly lower than that obtained in control cells kept in vitro at either 1.25 mmol/L or 0.8 mmol/L [Ca2+]e. In normal [Ca2+]e, the EGF-induced increases in [Ca2+]i were abolished by inhibiting EGF receptor autophosphorylation and by blocking calcium channels. Low in vitro [Ca2+]e significantly dampened the EGF-mediated DNA synthesis in normal rat hepatocytes but hypocalcemia in vivo further reduced the proliferative response compared with that obtained in control rat hepatocytes maintained in normal, or low [Ca2+]e. Furthermore, the blunted responses in [Ca2+]i mobilization and DNA synthesis associated with hypocalcemia could not be overcome by increasing concentrations of EGF nor by normalization of [Ca2+]e in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Calcium; Cell Division; Cells, Cultured; DNA; Dose-Response Relationship, Drug; Epidermal Growth Factor; Hypocalcemia; Kinetics; L-Lactate Dehydrogenase; Liver; Male; Rats; Rats, Sprague-Dawley; Time Factors

Several lines of evidence indicate that calcium deficiency is associated with cellular defects in many tissues and organs. Owing to the large in vivo gradient between ionized extra- and intracellular Ca2+ concentrations ([Ca2+]i), it is generally recognized that the prevailing circulating Ca2+ does not significantly affect resting cytosolic Ca2+. To probe the consequences of hypocalcemia on [Ca2+]i, a model of chronic hypocalcemia secondary to vitamin D (D) deficiency was used. Hepatocytes were isolated from livers of hypocalcemic D-deficient, of normocalcemic D3-repleted, or of normal control rats presenting serum Ca2+ of 0.78 +/- 0.02, 1.24 +/- 0.03, or 1.25 +/- 0.01 mM, respectively (P < 0.0001). [Ca2+]i was measured in cell couplets using the fluorescent probe Fura-2. Hepatocytes of normocalcemic D3-repleted and of normal controls exhibited similar [Ca2+]i of 227 +/- 10 and 242 +/- 9 nM, respectively (NS), whereas those of hypocalcemic rats had significantly lower resting [Ca2+]i (172 +/- 10 nM; P < 0.0003). Stimulation of hepatocytes with the alpha 1-adrenoreceptor agonist phenylephrine illicited increases in cytosolic Ca2+ leading to similar [Ca2+]i and phosphorylase a (a Ca(2+)-dependent enzyme) activity in all groups but in contrast to normocalcemia, low extracellular Ca2+ was often accompanied by a rapid decay in the sustained phase of the [Ca2+]i response. When stimulated with the powerful hepatic mitogen epidermal growth factor (EGF), hepatocytes isolated from hypocalcemic rat livers responded with a blunted maximal [Ca2+]i of 237.6 +/- 18.7 compared with 605.2 +/- 89.9 nM (P < 0.0001) for their normal counterparts, while the EGF-mediated DNA synthesis response was reduced by 50% by the hypocalcemic condition (P < 0.03). Further studies on the possible mechanisms involved in the perturbed [Ca2+]i homeostasis associated with chronic hypocalcemia revealed the presence of an unchanged plasma membrane Ca2+ ATPase but of a significant decrease in agonist-stimulated Ca2+ entry as indicated using Mn2+ as surrogate ion (P < 0.03). Our data, thus indicate that, in rat hepatocytes, the in vivo calcium status significantly affects resting [Ca2+]i, and from this we raise the hypothesis that this lower than normal [Ca2+]i may be linked, in calcium disorders, to inappropriate cell responses mediated through the calcium signaling pathway as illustrated by the response to phenylephrine and EGF.

Topics: Animals; Calcium; Cells, Cultured; Cholecalciferol; Epidermal Growth Factor; Homeostasis; Hypocalcemia; Liver; Male; Phenylephrine; Rats; Rats, Sprague-Dawley; Vitamin D Deficiency

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] is known to influence cell proliferation/maturation, whereas epidermal growth factor (EGF) is a potent stimulant of proliferation. Recently, hypocalcemia of vitamin D (D) deficiency was shown to significantly perturbe hepatic regeneration, which could be only partly restored by normalizing extracellular calcium, whereas normalization of 1,25-(OH)2D3 fully restored the process. To define the calcium- and/or D3-sensitive mechanisms associated with liver growth, a study of the initial events transduced by EGF was initiated by probing EGF receptor (EGFR) density and affinity, its subsequent autophosphorylation, and the level of its steady state transcript. Studies were carried out in D-depleted rats kept either untreated or supplemented with D3, 1,25-(OH)2D3, or calcium alone. The hepatic EGFR number (picomoles per mg microsomal protein) was significantly affected by hypocalcemic D-depleted (0.82 +/- 0.2), but responded with similar increases to calcium (1.7 +/- 0.09; P < 0.05), D3 (1.6 +/- 0.3; P < 0.05), and 1,25-(OH)2D3 (2.1 +/- 0.3; P < 0.01). The EGFR mRNA level revealed, however, no significant effect of the calcium or D3 status, indicating that posttranscriptional events were playing an important role. Phosphorylation studies showed that EGFR autophosphorylation and tyrosine protein kinase activity paralleled receptor density, with the lowest autophosphorylation values obtained in hypocalcemic D-depleted rats (D-depleted hypocalcemic vs. D3 repleted, P < 0.007). When normalized for receptor number, however, EGFR autophosphorylation increased in D-depleted hypocalcemic rats to a level comparable to that observed in all other groups. To dissociate the effect of the D3 hormone from that of calcium alone on EGFR, D-depleted rats were treated with the nonhypercalcemic 1,25-(OH)2D3 analog 22-OXA-1,25-(OH)2D3 (OCT), with or without calcium supplementation. Hypocalcemic OCT-treated rats did not exhibit any increase in EGFR number (0.6 +/- 0.1) compared to D-depleted hypocalcemic rats, but the addition of dietary calcium to OCT restored extracellular calcium concentrations and EGFR density (1.8 +/- 0.2; P < 0.002) to values comparable to those observed after D3 or 1,25-(OH)2D3 treatment. EGFR autophosphorylation was also decreased in hypocalcemic OCT-treated animals (P < 0.03), but after normalization for receptor density, full restoration of EGFR autophosphorylation was achieved. Our data demonstrate that in normal hepat

Topics: Animals; Calcitriol; Calcium; Cholecalciferol; Epidermal Growth Factor; ErbB Receptors; Female; Hypocalcemia; Liver; Male; Phosphorylation; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transforming Growth Factor alpha; Vitamin D Deficiency

During iv infusions of epidermal growth factor into sheep, serum calcium concentrations fell, whereas serum magnesium and serum immunoreactive PTH levels increased. Urinary calcium and magnesium decreased significantly. The role of epidermal growth factor in calcium homeostasis is discussed.

Topics: Animals; Calcium; Epidermal Growth Factor; Hypocalcemia; Magnesium; Male; Orchiectomy; Parathyroid Hormone; Sheep; Time Factors