CHRONIC HYPOCALCEMIA OF VITAMIN-D DEFICIENCY LEADS TO LOWER INTRACELLULAR CALCIUM CONCENTRATIONS IN RAT HEPATOCYTES

Several lines of evidence indicate that calcium deficiency is associat ed,vith cellular defects in many tissues and organs. Owing to the larg e in vivo gradient between ionized extra- and intracellular Ca2+ conce ntrations ([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 ch ronic hypocalcemia secondary to vitamin D (D) deficiency was used. Hep atocytes were isolated from livers of hypocalcemic D-deficient, of nor mocalcemic D-3-repleted, or of normal control rats presenting serum Ca 2+ 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 fluore scent probe Fura-2. Hepatocytes of normocalcemic D-3-repleted and of n ormal controls exhibited similar [Ca2+](i) of 227 +/- 10 and 242 +/- 9 nM, respectively (NS), whereas those of hypocalcemic rats had signifi cantly lower resting [Ca2+](i) (172 +/- 10 nM; P < 0.0003). Stimulatio n of hepatocytes with the alpha(1)-adrenoreceptor agonist phenylephrin e ilicited increases in cytosolic Ca2+ leading to similar [Ca2+](i) an d phosphorylase a (a Ca2+-dependent enzyme) activity in all groups but in contrast to normocalcemia, low extracellular Ca2+ was often accomp anied by a rapid decay in the sustained phase of the [Ca2+](i) respons e. When stimulated with the powerful hepatic mitogen epidermal growth factor (EGF), hepatocytes isolated from hypocalcemic rat livers respon ded with a blunted maximal [Ca2+](i) of 237.6 +/- 18.7 compared with 6 05.2 +/- 89.9 nM (P < 0.0001) for their normal counterparts, while the EGF-mediated DNA synthesis response was reduced by 50% by the hypocal cemic condition (P < 0.03). Further studies on the possible mechanisms involved in the perturbed [Ca2+](i) homeostasis associated with chron ic hypocalcemia revealed the presence of an unchanged plasma membrane Ca(2+)ATPase but of a significant decrease in agonist-stimulated Ca2entry as indicated using Mn2+ as surrogate ion (P < 0.03). Our data, t hus indicate that, in rat hepatocytes, the in vivo calcium status sign ificantly affects resting [Ca2+](i), and from this we raise the hypoth esis that this lower than normal [Ca2+](i) may be linked, in calcium d isorders, to inappropriate cell responses mediated through the calcium signaling pathway as illustrated by the response to phenylephrine and EGF.